Inhibitors of human plasmin derived from the kunitz domains

ABSTRACT

This invention provides: novel proteins, which are homologous to the first Kunitz domain (K1) of lipoprotein-associated coagulation inhibitor (LACI), and which are capable of inhibiting plasmin; uses of such novel proteins in therapeutic, diagnostic, and clinical methods; and polynucleotides that encode such novel proteins.

The present application is a continuation-in-part of application Ser.No. 08/208,265 (now pending) which in turn is a continuation-in-part ofSer. No. 08/179,658, filed Jan. 11, 1994. The entirety of each of theseapplications is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel mutants of the first Kunitz domain (K₁)of the human lipoprotein-associated coagulation inhibitor LACI, whichinhibit plasmin. The invention also relates to other modified Kunitzdomains that inhibit plasmin and to other plasmin inhibitors.

2. Description of the Background Art

The agent mainly responsible for fibrinolysis is plasmin, the activatedform of plasminogen. Many substances can activate plasminogen, includingactivated Hageman factor, streptokinase, urokinase (uPA), tissue-typeplasminogen activator (tPA), and plasma kallikrein (pKA). pKA is both anactivator of the zymogen form of urokinase and a direct plasminogenactivator.

Plasmin is undetectable in normal circulating blood, but plasminogen,the zymogen, is present at about 3 μM. An additional, unmeasured amountof plasminogen is bound to fibrin and other components of theextracellular matrix and cell surfaces. Normal blood contains thephysiological inhibitor of plasmin, α₂-plasmin inhibitor (α₂-PI), atabout 2 μM. Plasmin and α₂-PI form a 1:1 complex Matrix or cellbound-plasmin is relatively inaccessible to inhibition by α₂-PI. Thus,activation of plasmin can exceed the neutralizing capacity of α₂-PIcausing a profibrinolytic state.

Plasmin, once formed:

-   i. degrades fibrin clots, sometimes prematurely;-   ii. digests fibrinogen (the building material of clots) impairing    hemostasis by causing formation of friable, easily lysed clots from    the degradation products, and inhibition or platelet    adhesion/aggregation by the fibrinogen degradation products;-   iii. interacts directly with platelets to cleave glycoproteins Ib    and IIb/IIIa preventing adhesion to injured endothelium in areas of    high shear blood flow and impairing the aggregation response needed    for platelet plug formation (ADEL86);-   iv. proteolytically inactivates enzymes in the extrinsic coagulation    pathway further promoting a prolytic state.

Robbins (ROBB87) reviewed the plasminogen-plasmin system in detail.ROBB87 and references cited therein are hereby incorporated byreference.

Fibrinolysis and Fibrinogenolysis

Inappropriate fibrinolysis and fibrinogenolysis leading to excessivebleeding is a frequent complication of surgical procedures that requireextracorporeal circulation, such as cardiopulmonary bypass, and is alsoencountered in thrombolytic therapy and organ transplantation,particularly liver. Other clinical conditions characterized by highincidence of bleeding diathesis include liver cirrhosis, amyloidosis,acute promyelocytic leukemia, and solid tumors. Restoration ofhemostasis requires infusion of plasma and/or plasma products, whichrisks immunological reaction and exposure to pathogens, e.g. hepatitisvirus and HIV.

Very high blood loss can resist resolution even with massive infusion.When judged life-threatening, the hemorrhage is treated withantifibrinolytics such as ε-amino caproic acid (See HOOV93) (EACA),tranexamic acid, or aprotinin (NEUH89). Aprotinin is also known asTrasylol™ and as Bovine Pancreatic Trypsin Inhibitor (BPTI).Hereinafter, aprotinin will be referred to as “BPTI”. EACA andtranexamic acid only prevent plasmin from binding fibrin by binding thekringles, thus leaving plasmin as a free protease in plasma. BPTI is adirect inhibitor of plasmin and is the most effective of these agents.Due to the potential for thrombotic complications, renal toxicity and,in the case of BPTI, immunogenicity, these agents are used with cautionand usually reserved as a “last resort” (PUTT89). All three of theantifibrinolytic agents lack target specificity and affinity andinteract with tissues and organs through uncharacterized metabolicpathways. The large doses required due to low affinity, side effects dueto lack of specificity and potential for immune reaction andorgan/tissue toxicity augment against use of these antifibrinolyticsprophylactically to prevent bleeding or as a routine postoperativetherapy to avoid or reduce transfusion therapy. Thus, there is a needfor a safe antifibrinolytic. The essential attributes of such an agentare:

-   -   i. Neutralization of relevant target fibrinolytic enzyme(s);    -   ii. High affinity binding to target enzymes to minimize dose;    -   iii. High specificity for target, to reduce side effects; and    -   iv. High degree of similarity to human protein to minimize        potential immunogenicity and organ/tissue toxicity.        All of the fibrinolytic enzymes that are candidate targets for        inhibition by an efficacious antifibrinolytic are        chymotrypin-homologous serine proteases.

Excessive Bleeding

Excessive bleeding can result from deficient coagulation activity,elevated fibrinolytic activity, or a combination of the two conditions.In most bleeding diatheses one must control the activity of plasmin. Theclinically beneficial effect of BPTI in reducing blood loss is thoughtto result from its inhibition of plasmin (K_(D)˜0.3 nM) or of plasmakallikrein (K_(D)˜100 nM) or both enzymes.

GARD93 reviews currently-used thrombolytics, saying that, althoughthrombolytic agents (e.g. tPA) do open blood vessels, excessive bleedingis a serious safety issue. Although tPA and streptokinase have shortplasma half lives, the plasmin they activate remains in the system for along time and, as stated, the system is potentially deficient in plasmininhibitors. Thus, excessive activation of plasminogen can lead to adangerous inability to clot and injurious or fatal hemorrhage. A potent,highly specific plasmin inhibitor would be useful in such cases.

BPTI is a potent plasmin inhibitor; it has been found, however, that itis sufficiently antigenic that second uses require skin testing.Furthermore, the doses of BPTI required to control bleeding are quitehigh and the mechanism of action is not clear. Some say that BPTI actson plasmin while others say that it acts by inhibiting plasmakallikrein. FRAE89 reports that doses of about 840 mg of BPTI to 80open-heart surgery patients reduced blood loss by almost half and themean amount transfused was decreased by 74%. Miles Inc. has recentlyintroduced Trasylol in USA for reduction of bleeding in surgery (SeeMiles product brochure on Trasylol, which is hereby incorporated byreference.) LOHM93 suggests that plasmin inhibitors may be useful incontrolling bleeding in surgery of the eye. SHER89 reports that BPTI maybe useful in limiting bleeding in colonic surgery.

A plasmin inhibitor that is approximately as potent as BPTI or morepotent but that is almost identical to a human protein domain offerssimilar therapeutic potential but poses less potential for antigenicity.

Angiogenesis:

Plasmin is the key enzyme in angiogenesis. OREI94 reports that a 38 kDafragment of plasmin (lacking the catalytic domain) is a potent inhibitorof metastasis, indicating that inhibition of plasmin could be useful inblocking metastasis of tumors (FIDL94). See also ELLI92. ELLI92, OREI94and FIDL94 and the references cited there are hereby incorporated byreference.

Plasmin

Plasmin is a serine protease derived from plasminogen. The catalyticdomain of plasmin (or “CatDom”) cuts peptide bonds, particularly afterarginine residues and to a lesser extent after lysines and is highlyhomologous to trypsin, chymotrypsin, kallikrein, and many other serineproteases. Most of the specificity of plasmin derives from the kringles'binding of fibrin (LUCA83, VARA83, VARA84). On activation, the bondbetween ARG₅₆₁-Val₅₆₂ is cut, allowing the newly free amino terminus toform a salt bridge. The kringles remain, nevertheless, attached to theCatDom through two disulfides (COLM87, ROBB87).

BPTI has been reported to inhibit plasmin with K_(D) of about 300 pM(SCHN86). AUER88 reports that BPTI(R₁₅) has K_(i) for plasmin of about13 nM, suggesting that R₁₅ is substantially worse than K₁₅ for plasminbinding. SCHN86 reports that BPTI in which the residues C₁₄ and C₃₈ havebeen converted to Alanine has K_(i) for plasmin of about 4.5 nM. KIDO88reports that APP-I has K_(i) for plasmin of about 75 pM (7.5×10⁻¹¹ M),the most potent inhibitor of human plasmin reported so far. DENN94areports, however, that APP-I inhibits plasmin with K_(i)=225 nM(2.25×10⁻⁷ M). Our second and third library were designed under theassumption that APP-I is a potent plasmin binder. The selection processdid not select APP-I residues at most locations and the report ofDENN94a explains why this happened.

With recombinant DNA techniques, it is possible to obtain a novelprotein by expressing a mutated gene encoding a mutant of the nativeprotein gene. Several strategies for picking mutations are known. In onestrategy, some residues are kept constant, others are randomly mutated,and still others are mutated in a predetermined manner. This is called“variegation” and is defined in Ladner el al. U.S. Pat. No. 5,223,409,which is incorporated by reference.

DENN94a and DENN94b report selections of Kunitz domains based on APP-Ifor binding to the complex of Tissue Factor with Factor VII_(a). Theydid not use LACI-K1 as parental and did not use plasmin as a target. Thehighest affinity binder they obtained had K_(D) for their target ofabout 2 nM. Our first-round selectants have affinity in this range, butour second round selectants are about 25-fold better than this.

Proteins taken from a particular species are assumed to be less likelyto cause an immune response when injected into individuals of thatspecies. Murine antibodies are highly antigenic in humans. “Chimeric”antibodies having human constant domains and murine variable domains aredecidedly less antigenic. So called “humanized” antibodies have humanconstant domains and variable domains in which the CDRs are taken frommurine antibodies while the framework of the variable domains are ofhuman origin. “Humanized” antibodies are much less antigenic than are“chimeric” antibodies. In a “humanized” antibody, fifty to sixtyresidues of the protein are of non-human origin. The proteins of thisinvention comprise, in most cases, only about sixty amino acids andusually there are ten or fewer differences between the engineeredprotein and the parental protein. Although humans do develop antibodieseven to human proteins, such as human insulin, such antibodies tend tobind weakly and the often do not prevent the injected protein fromdisplaying its intended biological function. Using a protein from thespecies to be treated does not guarantee that there will be no immuneresponse. Nevertheless, picking a protein very close in sequence to ahuman protein greatly reduces the risk of strong immune response inhumans.

Kunitz domains are highly stable and can be produced efficiently inyeast or other host organisms. At least ten human Kunitz domains havebeen reported. Although APP-I was thought at one time to be a potentplasmin inhibitor, there are, actually, no human Kunitz domains thatinhibit plasmin as well as does BPTI. Thus, it is a goal of the presentinvention to provide sequences of Kunitz domain that are both potentinhibitors of plasmin and close in sequence to human Kunitz domains.

The use of site-specific mutagenesis, whether nonrandom or random, toobtain mutant binding proteins of improved activity is known in the art,but success is not assured.

SUMMARY OF THE INVENTION

This invention relates to mutants of BPTI-homologous Kunitz domains thatpotently inhibit human plasmin. In particular, this invention relates tomutants of one domain of human LACI which are likely to benon-immunogenic to humans, and which inhibit plasmin with K_(D),preferably, of about 5 nM or less, more preferably of about 300 pM orless, and most preferably about 100 pM or less. The invention alsorelates to the therapeutic and diagnostic use of these novel proteins.

Plasmin-inhibiting proteins are useful for the prevention or treatmentof clinical conditions caused or exacerbated by plasmin, includinginappropriate fibrinolysis or fibrinogenolysis, excessive bleedingassociated with thrombolytics, post-operative bleeding, andinappropriate androgenesis. Plasmin-binding mutants, whether or notinhibitory, are useful for assaying plasmin in samples, in vitro, forimaging areas of plasmin activity, in vivo, and for purification ofplasmin.

Preferred mutants QS4 and NS4 were selected from a library that allowedabout 50 million proteins having variability at positions 13, 16, 17,18, 19, 31, 32, 34, and 39. These proteins have an amino-acid sequencenearly identical to a human protein but inhibit plasmin with K_(i) ofabout 2 nM (i.e. about 6-fold less potent than BPTI, but 100-fold betterthan APP-I).

An especially preferred protein, SPI11, was selected from a libraryallowing variability at positions 10, 11, 13, 15, 16, 17, 18, 19, and 21and has an affinity for plasmin which is less than 100 μM (i.e. about3-fold superior to BPTI in binding), and yet is much more similar insequence to LACI, a human protein, than to the BPTI, a bovine protein.Other LACI-K1 mutants selected from this library and thought to havevery high affinity for plasmin include SPI51, SPI08, and SPI23. Anadditional library allowing variation at positions 10, 11, 13, 15, 16,17, 18, 19, 21, 31, 32, 34, 35, and 39 has been screened and a consensussequence (SPIcon1) found. Variants shown to be better than QS4, and thusmore preferred, include SPI51 and SPI47. Sequences that are likely tohave very high affinity for plasmin yet retain an essentially humanamino-acid sequence have been identified and include sequences SPI60,SPI59, SPI42, SPI55, SPI56, SPI52, SPI46, SPI49, SPI53, SPI41, andSPI57. The amino-acid sequence information that confers high affinityfor the active site of plasmin can be transferred to other Kunitzdomains, particularly to Kunitz domains of human origin; designs ofseveral such proteins are disclosed.

The preferred plasmin inhibitors of the present invention fulfill one ormore of the following desiderata:

-   1) the K_(i) for plasmin is at most 20 nM, preferably not more than    about 5 nM, more preferably not more than about 300 pM, and most    preferably, not more than about 100 pM,-   2) the inhibitor comprises a Kunitz domain meeting the requirements    shown in Table 14 with residues number by reference to BPTI,-   3) at the Kunitz domain positions 12-21 and 32-39 one of the    amino-acid types listed for that position-in Table 15, and-   4) the inhibitor is more similar in amino-acid sequence to a    reference sequence selected from the group SPI11, SPI15, SPI08,    SPI23, SPI51, SPI47, QS4, NS4, Human LACI-K2, Human LACI-K3, Human    collagen α3 KuDom, Human TFPI-2 DOMAIN 1, Human TFPI-2 DOMAIN 2,    Human TFPI-2 DOMAIN 3, HUMAN ITI-K1, Human ITI-K2, HUMAN PROTEASE    NEXIN-II, Human APP-I, DPI-1.1.1, DPI-1.1.2, DPI-1.1.3, DPI-1.2.1,    DPI-1.3.1, DPI-2.1, DPI-3.1.1, DPI-3.2.1, DPI-3.3.1, DPI-4.1.1,    DPI-4.2.1, DPI-4.2.2, DPI-4.2.3, DPI-4.2.4, DPI-4.2.5, DPI-5.1,    DPI-5.2, DPI-6.1, DPI-6.2 than is the amino acid sequence of said    Kunitz domain to the sequence of BPTI.

Nomenclature

Herein, affinities are stated as K_(D)(K_(D)(A, B)=[A][B]/[A−B]). Anumerically smaller K_(D) reflects higher affinity. For the purposes ofthis invention, a “plasmin inhibiting protein” is one that binds andinhibits plasmin with K_(i) of about 20 nM or less. “Inhibition” refersto blocking the catalytic activity of plasmin and so is measurable invitro in assays using chromogenic or fluorogenic substrates or in assaysinvolving macromolecules.

Amino-acid residues are discussed in three ways: full name of the aminoacid, standard three-letter code, and standard single-letter code. Tableuse only the one-letter code. The text uses full names and three-lettercode where clarity requires.

A = Ala C = Cys D = Asp E = Glu F = Phe G = Gly H = His I = Ile K = LysL = Leu M = Met N = Asn P = Pro Q = Gln R = Arg S = Ser T = Thr V = ValW = Trp Y = Tyr

For the purposed of this invention, “substantially homologous” sequencesare at least 51%, mnore preferably at least 80%, identical, over anyspecifiied regions. Herein, sequences that are identical are understoodto be “substantially homologous”. Sequences would still be“substantially homologous” if within one region of at least 20 aminoacids they are sufficiently similar (51% or more) but outside the regionof comparison they differed totally. An insertion of one amino acid inone sequence relative to the other counts as one mismatch. Mostpreferably, no more than six residues, other than at termini, aredifferent. Preferably, the divergence in sequence, particularly in thespecified regions, is in the form of “conservative modifications”.

“Conservative modifications” are defined as

-   (a) conservative substitutions of amino acids as defined in Table 9;    and-   (b) single or multiple insertions or deletions of amino acids at    termini, at domain boundaries, in loops, or in other segments of    relatively high mobility.

Preferably, except at termini, no more than about six amino acids areinserted or deleted at any locus, and the modifications are outsideregions known to contain important binding sites.

Kunitz Domains

Herein, “Kunitz domain” and “KuDom” are used interchangeably to mean ahomologue of BPTI (not of the Kunitz soya-bean trypsin inhibitor). AKuDom is a domain of a protein having at least 51 amino acids (and up toabout 61 amino acids) containing at least two, and preferably three,disulfides. Herein, the residues of all Kunitz domains are numbered byreference to BPTI (i.e. residues 1-58). Thus the first cysteine residueis residue 5 and the last cysteine is 55. An amino-acid sequence shall,for the purposed of this invention, be deemed a Kunitz domain if it canbe aligned, with three or fewer mismatches, to the sequence shown inTable 14. An insertion or deletion of one residue shall count as onemismatch In Table 14, “x” matches any amino acid and “X” matches thetypes listed for that position. Disulfides bonds link at least two of: 5to 55, 14 to 38, and 30 to 51. The number of disulfides may be reducedby one, but none of the standard cysteines shall be left unpaired. Thus,if one cysteine is changed, then a compensating cysteine is added in asuitable location or the matching cysteine is also replaced by anon-cysteine (the latter being generally preferred). For example,Drosophila funebris male accessory gland protease inhibitor has nocysteine at position 5, but has a cysteine at position −1 Gust beforeposition 1); presumably this forms a disulfide to CYS₅₅. If Cys₁₄ andCys₃₈ are replaced, the requirement of Gly₁₂, (Gly or Ser)₃₇, and Gly₃₆are dropped. From zero to many residues, including additional domains(including other KuDoms), can be attached to either end of a Kunitzdomain.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Protease inhibitors, such as Kunitz domains, function by binding intothe active site of the protease so that a peptide bond (the “scissilebond”) is: 1) not cleaved, 2) cleaved very slowly, or 3) cleaved to noeffect because the structure of the inhibitor prevents release orseparation of the cleaved segments. In Kunitz domains, disulfide bondsact to hold the protein together even if exposed peptide bonds arecleaved. From the residue on the amino side of the scissile bond, andmoving away from the bond, residues are conventionally called P1, P2,P3, etc. Residues that follow the scissile bond are called P1′, P2′,P3′, etc. (SCHE67, SCHE68). It is generally accepted that each serineprotease has sites (comprising several residues) S1, S2, etc. thatreceive the side groups and main-chain atoms of residues P1, P2, etc. ofthe substrate or inhibitor and sites S1′, S2′, etc. that receive theside groups and main-chain atoms of P1′, P2′, etc. of the substrate orinhibitor. It is the interactions between the S sites and the P sidegroups and main chain atoms that give the protease specificity withrespect to substrates and the inhibitors specificity with respect toproteases. Because the fragment having the new amino terminus leaves theprotease first, many worker designing small molecule protease inhibitorshave concentrated on compounds that bind sites S1, S2, S3, etc.

LASK80 reviews protein protease inhibitors. Some inhibitors have severalreactive sites on one polypeptide chains and these domains usually havedifferent sequences, specificities, and even topologies. It is knownthat substituting amino acids in the P₅ to P₅′ region influences thespecificity of an inhibitor. Previously, attention has been focused onthe P1 residue and those very dose to it because these can change thespecificity from one enzyme class to another. LASK80 suggests that amongKuDoms, inhibitors with P1=Lys or Arg inhibit trypsin, those withP1=Tyr, Phe, Trp, Leu and Met inhibit chymotrypsin, and those withP1=Ala or Ser are likely to inhibit elastase. Among the Kazalinhibitors, LASK80 continues, inhibitors with P1=Leu or Met are stronginhibitors of elastase, and in the Bowman-Kirk family elastase isinhibited with P1=Ala, but not with P1=Leu. Such limited changes do notprovide inhibitors of truly high affinity (i.e. better than 1 to 10 nM).

Kunitz domains are defined above. The 3D structure (at high resolution)of BPTI (the archetypal Kunitz domain) is known. One of the X-raystructures is deposited in the Brookhaven Protein Data Bank as “6PTI”].The 3D structure of some BPTI homologues (EIGE90, HYNE90) are known. Atleast seventy KuDom sequences are known. Known human homologues includethree KuDoms of LACI (WUNT88, GIRA89, NOVO89), two KuDoms ofInter-α-Trypsin Inhibitor, APP-I (KIDO88), a KuDom from collagen, andthree KuDoms of TFPI-2 (SPRE94).

LACI

Lipoprotein-associated coagulation inhibitor (LACI) is a human serumphosphoglycoprotein with a molecular weight of 39 kDa (amino-acidsequence in Table 1) containing three KuDoms. We refer hereinafter tothe protein as LACI and to the Kunitz domains thereof as LACI-K1(residues 50 to 107), LACI-K2 (residues 121 to 178), and LACI-K3 (213 to270). The cDNA sequence of LACI is reported in WUNT88. GIRA89 reportsmutational studies in which the P1 residues of each of the three KuDomswere altered. LACI-K1 inhibits Factor VIIa (F.VII_(a)) when F.VII_(a) iscomplexed to tissue factor and LACI-K2 inhibits Factor X_(a). It is notknown whether LACI-K3 inhibits anything. Neither LACI nor any of theKuDoms of LACI is a potent plasmin inhibitor.

KuDoms of this invention are substantially homologous with LACI-K1, butdiffer in ways that confer strong plasmin inhibitory activity discussedbelow. Other KuDoms of this invention are homologous to othernaturally-occurring KuDoms, particularly to other human KuDoms. For usein humans, the proteins of this invention are designed to be moresimilar in sequence to a human KuDom than to BPTI, to reduce the risk ofcausing an immune response.

First Library of LACI-K1 and Selectants for Binding to Plasmin

Applicants have screened a first library of LACI-K1 for mutants havinghigh affinity for human plasmin and obtained the sequences shown inTable 2 and Table 3. These sequences may be summarized as shown in Table16, where “preferred residues” are those appearing in at least one ofthe 32 variants identified as binding plasmin. The preferences atresidues 13, 16, 17, 18 and 19 are strong, as shown in Table 17.Although the range of types allowed at 31 and 32 is limited, theselection indicates that an acidic group at 31 and a neutral group at 32is preferred. At residue 17, Arg was preferred; Lys, another positivelycharged amino acid, was not in the library, and may be a suitablesubstitute for Arg. Many amino-acid types at positions 34 and 39 areconsistent with high-affinity plasmin binding, but some types may hinderbinding.

It should be appreciated that Applicants have not sequenced all thepositive isolates of this or other libraries herein disclosed, and thatsome of the possible proteins may not have been present in detectableamounts.

Applicants have prepared one of the selected proteins, QS4, shown inTable 2. QS4 inhibits plasmin with a K_(i) of about 2 nM. Although thislevel of inhibition is less than that of BPTI, QS4 is a preferredmolecule for use in humans because it has less potential forimmunogenicity. Other proteins shown in Table 2 and Table 3 are verylikely to be potent inhibitors of plasmin and are likely to pose littlethreat of antigenicity.

Second Library that Varies Residues 10-21

Applicants have prepared a second library of LACI-K1 derivatives shownin Table 5 and allowing variation at residues 10, 11, 13, 15, 16, 17,18, 19, and 21. This was screened for binding to plasmin and theproteins shown in Table 6 were obtained.

“Consensus” in Table 6 is E ₁₀ TGPCRARFERW ₂₁, where the sevenunderscored residues differ from LACI-K1. Only acidic amino acids(Glu:17 or Asp:15) were seen at position 10; Lys and Asn are notacceptable. As Glu and Asp appeared with almost equal frequency, theyprobably to contribute equally to binding. Acidic residues were not seenat position 11. Thr was most common (11/32) with Ser appearing often(9/32); Gly appeared 8 times. At 13, Pro was strongly preferred (24/32)with Ala second at 5/32. At 15, Arg was strongly preferred (25/32), buta few (7/32) isolates have Lys. Note that BPTI(R₁₅) is a worse plasmininhibitor than is BPTI. At 16, Ala was preferred (22/32), but Gly didappeared fairly often (10/32). At 17, Arg was most common (15/32), withLys coming second (9/32). At residues 17 and 18, APP-I has Met and Ile.At 18, we allowed Ile or Phe. Only four isolates have Ile at 18 and noneof these have Met at 17. This was surprising in view of KIDO88, butquite understandable in view of DENN94a. This collection of isolates hasa broad distribution at 19: (Glu:8, Pro:7, Asp:4, Ala:3, His:3, Gly:2,Gln:2, Asn:1, Ser:1, and Arg:1), but acidic side groups are stronglypreferred over basic ones. At 21, the distribution was (Trp:16, Phe:14,Leu:2, Cys:0); BPTI has Tyr at 21.

The binding of clonally pure phage that display one or another of theseproteins was compared to the binding of BPTI phage (Table 6). Applicantshave determined the K_(i) of protein SPI11 and found it to be about 88pM which is substantially superior to BPTI.

Third Library that Varies 10-21 and 31-39

Applicants used a pool of phage of the second library (varied atresidues 10, 11, 13, 15, 16, 17, 18, 19, and 21) that had been selectedtwice for plasmin binding as a source of DNA into which variegation wasintroduced at residues 31, 32, 34, 35, and 39 as shown in Table 7.

This library was screened for three rounds for binding to plasmin andthe isolates shown in Table 8 were obtained. The distribution ofamino-acid types is shown in Table 18 where “x” means the amino-acidtype was not allowed and “*” indicates the wild-type for LACI-K1.

These sequences gave a consensus in the 10-21 and 31-40 region of E ₁₀TGPCRAKFDRW ₂₁ . . . E₃₁ AFVYGGCGG₄₀ (SPIcon1 in Table 4). The tenunderscored amino acids differ from LACI-K1. At eight varied positions,a second type was quite common: Asp at 10, Ala at 11, Glu at 19, Phe at21, Thr at 31, Pro or Ser at 32, Leu or Ile at 34, and Glu at 39. Atposition 17, the highly potent inhibitor SPI11 has R. Thus, the sequenceD₁₀ TGPCRARFDRF₂₁ . . . E₃₁ AFTYGGCEG₄₀ (DPI-1.1.1 in Table 4) differsfrom LACI-K1 by only six residues, matches the selected sequences at theresidues having strong consensus, and has preferred substitutions atpositions 10, 17, 21, 34, and 39. DPI-1.1.1. is expected to have a veryhigh affinity for plasmin and little potential for immunogenicity inhumans.

Preliminary testing of proteins SPI11, BPTI, SPI23, SPI51, SPI47, QS4,SPI22, SPI54, and SPI43 for plasmin inhibitory activity placed them inthe order given. SPI11 is significantly more potent than BPTI with K_(i)of about 88 pM. SPI23 and SPI51 are very similar in activity and onlyslightly less potent than BPTI. SPI47 is less potent than SPI51 butbetter than QS4. SPI22 is weaker than QS4. SPI54 and SPI43 are not sopotent as QS4, K_(i) probably >4 nM.

A KuDom that is highly homologous at residues 5-55 to any one of thesequences SPI11. SPI15, SPI08, SPI23, SPI51, SPI47. QS4, and NS4, asshown in Table 4, is likely to be a potent inhibitor (K_(D)>5 nM) ofplasmin and have a low potential for antigenicity in humans. Morepreferably, to have high affinity for plasmin, a KuDom would have asequence that is identical at residues 10-21 and 31-39 and has five orfewer differences at residues 5-9, 22-30, and 40-55 as compared to anyof the sequences SPI11, SPI15, SPI08, SPI23, SPI51, SPI47, QS4, and NS4.

Using the selected sequences and the binding data of selected andnatural KuDoms, we can write a recipe for a high-affinityplasmin-inhibiting KuDom that can be applied to other human KuDomparentals. First, the KuDom must meet the requirements in Table 14. Thesubstitutions shown in Table 15 are likely to confer high-affinityplasmin inhibitory activity on any KuDom. Thus a protein that contains asequence that is a KuDom, as shown in Table 14, and that contains ateach of the position 12-21 and 32-39 an amino-acid type shown in Table15 for that position is likely to be a potent inhibitor of humanplasmin. More preferably, the protein would have an amino-acid typeshown in Table 15 for all of the positions listed in Table 15. To reducethe potential for immune response, one should use one or another humanKuDom as parental protein to give the sequence outside the bindingregion.

It is likely that a protein that comprises an amino-acid sequence thatis substantially homologous to SPI11 from residue 5 through residue 55(as shown in Table 4) and is identical to SPI11 at positions 13-19, 31,32, 34, and 39 will inhibit human plasmin with a K_(i) of 5 nM or less.SPI11 differs from LACI-K1 at 7 positions. It is not clear that thesesubstitutions are equally important in fostering plasmin binding andinhibition. There are seven molecules in which one of the substitutedpositions of SPI11 is changed to the residue found in LACI-K1 (i.e.“reverted”), 21 in which two of the residues are reverted, 35 in whichthree residues are reverted, 35 in which four are reverted, 21 in whichfive are reverted, and seven in which six are reverted. It is expectedthat those with more residues reverted will have less affinity forplasmin but also less potential for immunogenicity. A person skilled inthe art can pick a protein of sufficient potency and low immunogenicityfrom this collection of 126. It is also possible that substitutions inSPI11 by amino acids that differ from LACI-K1 can reduce theimmunogenicity without reducing the affinity for plasmin to a degreethat makes the protein unsuitable for use as a drug.

Designed KuDom Plasmin Inhibitors

Hereinafter, “DPI” will mean a “Designed Plasmin Inhibitor” that areKuDoms that incorporate amino-acid sequence information from the SPIseries of molecules, especially SPI11. Sequences of several DPIs andtheir parental proteins are given in Table 4.

Sequences DPI-1.1.1, DPI-1.1.2, DPI-1.1.3, DPI-1.1.4, DPI-1.1.5, andDPI-1.1.6 (in Table 4) differ from LACI-K1 by 6, 5, 5, 4, 3, and 2 aminoacids respectively and represent a series in which affinity for plasminmay decrease slowly while similarity to a human sequence increases so asto reduce likelihood of immunogenicity. The selections from each of thelibraries show that M18F is a key substitution and that either I17K orI17R is very important. Selections from the second and third libraryindicate that Arg is strongly preferred at 15, that an acid side groupat 11 is disadvantageous to binding. The highly potent inhibitor SPI11differs from the consensus by having R₁₇, as does BPTI. DPI-1.1.1carries the mutations D11T, K15R, I17R, M18F, K19D, and E32A, and islikely to be highly potent as a plasmin inhibitor. DPI-1.1.2 carriesD11T, K15R, I17R, M18F, and K19D, and is likely to be highly potent.DPI-1.1.3 carries the mutations D11A, K15R, I17R, M18F, and K19Drelative to LACI-K1. DPI-1.1.3 differs from DPI-1.1.2 by having A₁₁instead of T₁₁; both proteins are likely to be very potent plasmininhibitors. DPI-1.1.4 carries the mutations I17R M18F, K19D, and E32Aand should be quite potent. As DPI-1.1.4 has fewer of the SPI11mutations, it may be less potent, but is also less likely to beimmunogenic. DPI-1.1.5 carries the mutations I17R, M18F, and K19D). Thisprotein is likely to be a good inhibitor and is less likely to beimmunogenic. DPI-1.1.6 carries only the mutations I17R and M18F butshould inhibit plasmin.

Protein DPI-1.2.1 is based on human LACI-K2 and shown in Table 4. Themutations P11T, I13P, Y17R, I18F, T19D, R32E, K34I, and L39E are likelyto confer high affinity for plasmin. Some of these substitutions may notbe necessary; in particular, P11T and T19D may not be necessary. Othermutations that might improve the plasmin affinity include E9A, D10E,G16A, Y21W, Y21F, R32T, K34V, and L39G.

Protein DPI-1.3.1 (Table 4) is based on human LACI-K3. The mutationsR11T, L13P, N17R, E18F, N19D, R31E, P32E, K34I, and S36G are intended toconfer high affinity for plasmin. Some of these substitutions may not benecessary; in particular, N19D and P32E may not be necessary. Otherchanges that might improve K_(D) include D10E, N17K, F21W and G39E.

Protein DPI-2.1 (Table 4) is a based on the human collagen α3 KuDom. Themutations E11T, T13P, D16A, F17R, I18F, L19D, A31E, R32E, and W34I arelikely to confer high affinity for plasmin. Some of these substitutionsmay not be necessary; in particular, L19D and A31E may not be necessary.Other mutations that might improve the plasmin affinity include K9A,D10E, D16G, K20R, R32T, W34V, and G39E.

DPI-3.1.1 (Table 4) is derived from Human TFPI-2 domain 1. The exchangesY11T, L17R, L18F, L19D, and R31E are likely to confer high affinity forplasmin. The mutation L19D may not be needed. Other mutations that mightfoster plasmin binding include Y21W, Y21F, Q32E, L34I, L34V, and E39G.

DPI-3.2.1 (Table 4) is derived from Human TFPI-2 domain 2. This parentaldomain contains insertions after residue 9 (one residue) and 42 (tworesidues). The mutations (V₉SVDDQC₁₄ replaced by V₉ETGPC₁₄) E15R, S17K,T18F, K32T, F34V, and (H₃₉RNRIENR₄₄ replaced by (E₃₉GNRNR₄₄) are likelyto confer affinity for plasmin. Because of the need to change the numberof amino acids, DPI-3.2.1 has a higher potential for immunogenicity thando other modified human KuDoms.

DPI-3.3.1 (Table 4) is derived from human TFPI-2, domain 3. Thesubstitutions E11T, L13P, S15R, N17R, V18F, T34I, and T36G are likely toconfer high affinity for plasmin. The mutations E11T, L13P, and T34I maynot be necessary. Other mutations that might foster plasmin bindinginclude D10E, T19D, Y21W, and G39E.

DPI-4.1.1 (Table 4) is from human ITI-K1 by assertion of S10E, M15R,M17K, T18F, Q34V, and M39G. The mutations M39G and Q34V may not benecessary. Other mutations that should foster plasmin binding include:A11T, G16A, M17R, S19D, Y21W, and Y21F.

DPI-4.2.1 (Table 4) is from human ITI-K2 through the mutations V10D,R11T, F17R, I18F, and P34V. The mutation P34V might not be necessary.Other mutation that should foster plasmin binding include: V10E, Q19D,L20R, W21F, P34I, and Q39E. DPI-4.2.2 is an especially preferred proteinas it has only three mutations: R11T, F17R, and l18F. DPI-4.2.3 is anespecially preferred protein as it has only four mutations: R11T, F17R,I18F, and L20R. DPI-4.2.4 is an especially preferred protein as it hasonly five mutations: R11T, F17R, I18F, L20R, and P34V. DPI-4.2.5 carriesthe mutations V10E, R11T, F17R, I18F, L20R, V31E, L32T, P34V, and Q39Gand is highly likely to inhibit plasmin very potently. Each of theproteins DPI-4.2.1, DPI-4.2.2, DPI-4.2.3, DPI-4.2.4, and DPI-4.2.5 isvery likely to be a highly potent inhibitor of plasmin.

Before DENN94a, it was thought that APP-I was a very potent plasmininhibitor. Thus, it was surprising to select proteins from a librarythat was designed to allow the APP-I residues at positions 10-21 whichdiffered strongly from APP-I. Nevertheless, APP-I can be converted intoa potent plasmin inhibitor. DPI-5.1 is derived from human APP-I (alsoknown as Protease Nexin-II) by mutations M17R and I18F and is likely tobe a much better plasmin inhibitor than is APP-I itself DPI-5.2 carriesthe further mutations S19D, A31E, and F34I which may foster higheraffinity for plasmin.

DPI-6.1 is derived from the HKI B9 KuDom (NORR93) by the fivesubstitutions: K11T, Q15R, T16A, M17R, and M18F. DPI-6.1 is likely to bea potent plasmin inhibitor. DPI-6.2 carries the additional mutationsT19D and A34V which should foster plasmin binding.

Although BPTI is the best naturally-occurring KuDom plasmin inhibitorsknown, it could be improved. DPI-7.1 is derived from BPTI by themutation I18F which is likely to increase the affinity for plasmin.DPI-7.2 carries the further mutation K15R which should increase plasminbinding. DPI-7.3 carries the added mutation R39G. DPI-7.4 carries themutations Y10D, K15R, I18F, I19D, Q31E, and R39G and should have a veryhigh affinity for plasmin.

Modification of Kunitz Domains

KuDoms are quite small; if this should cause a pharmacological problem,such as excessively quick elimination from circulation, two or more suchdomains may be joined. A preferred linker is a sequence of one or moreamino acids. A preferred linker is one found between repeated domains ofa human protein, especially the linkers found in human BPTI homologues,one of which has two domains (BALD85, ALBR83b) and another of which hasthree (WUNT88). Peptide linkers have the advantage that the entireprotein may then be expressed by recombinant DNA techniques. It is alsopossible to use a nonpeptidyl linker, such as one of those commonly usedto form immunogenic conjugates. An alternative means of increasing theserum residence of a BPTI-like KuDom is to link it topolyethyleneglycol, so called PEGylation (DAVI79).

Ways to Improve Specificity of SPI11 and other KuDom Plasmin Inhibitors:

Because we have made a large part of the surface of the KuDom SPI11complementary to the surface of plasmin. R₁₅ is not essential forspecific binding to plasmin. Many of the enzymes in the clotting andfibrinolytic pathways cut preferentially after Arg or Lys. Not having abasic residue at the P1 position may give rise to greater specificity.The variant SPI11-R15A (shown in Table 11), having an ALA at P1, islikely to be a good plasmin inhibitor and may have higher specificityfor plasmin relative to other proteases than does SPI11. The affinity ofSPI11-R15A for plasmin is likely to be less than the affinity of SPI11for plasmin, but the loss of affinity for other Arg/Lys-preferringenzymes is likely to be greater and, in many applications, specificityis more important than affinity. Other mutants that are likely to havegood affinity and very high specificity include SPI11-R15G andSPI11-R15N-E32A. This approach could be applied to other high-affinityplasmin inhibitors.

Increasing the Affinity of SPI11

Variation of SPI11 as shown in Table 12 and selection of binders islikely to produce a Kunitz domain having affinity for plasmin that ishigher than SPI11. This fourth library allows variegation of the 14-38disulfide. The two segments of DNA shown are synthesized and used withprimers in a PCR reaction to produce ds DNA that runs from NsiI toBstEII. The primers are identical to the 5′ ends of the synthetic bitsshown and of length 21 for the first and 17 for the second. As thevariability is very high, we would endeavor to obtain between 10⁸ and10⁹ transformants (the more the better).

Mode of Production

Proteins of this invention may be produced by any conventionaltechnique, including

-   a) nonbiological synthesis by sequential coupling of components,    e.g. amino acids,-   b) production by recombinant DNA techniques in suitable host cells,    and-   c) semisynthesis, for example, by removal of undesired sequences    from LACI-K1 and coupling of synthetic replacement sequences.

Proteins disclosed herein are preferably produced, recombinantly, in asuitable host, such as bacteria from the genera Bacillus, Escherichia,Salmonella, Erwinia, and yeasts from the genera Hansenula,Kluyveromyces, Pichia, Rhinosporidium, Saccharomyces, andSchizosaccharomyces, or cultured mammalian cells such as COS-1. The morepreferred hosts are microorganisms of the species Pichia pastoris,Bacillus subtilis, Bacillus brevis, Saccharomyces cerevisiae,Escherichia coli and Yarrowia lipolytica. Any promoter which isfunctional in the host cell may be used to control gene expression.

Preferably the proteins are secreted and, most preferably, are obtainedfrom conditioned medium. Secretion is the preferred route becauseproteins are more likely to fold correctly and can be produced inconditioned medium with few contaminants. Secretion is not required.

Unless there is a specific reason to include glycogroups, we preferproteins designed to lack N-linked glycosylation sites to reducepotential for antigenicity of glycogroups and so that equivalentproteins can be expressed in a wide variety of organisms including: 1)E. coli, 2) B. subtilis, 3) P. pastoris, 4) S. cerevisiae, and 5)mammalian cells.

Several means exist for reducing the problem of host cells producingproteases that degrade the recombinant product; see, inter alia BANE90and BANE91. VAND92 reports that overexpression of the B. subtilis signalpeptidase in E. coli. leads to increased expression of a heterologousfusion protein. ANBA88 reports that addition of PMSF (a serine proteasesinhibitor) to the culture medium improved the yield of a fusion protein.

Other factors that may affect production of these and other proteinsdisclosed here include: 1) codon usage (optimizing codons for the hostis preferred), 2) signal sequence, amino-acid sequence at intendedprocessing sites, presence and localization of processing enzymes,deletion, mutation, or inhibition of various enzymes that might alter ordegrade the engineered product and mutations that make the host morepermissive in secretion (permissive secretion hosts are preferred).

Reference works on the general principles of recombinant DNA technologyinclude Watson et al., Molecular Biology of the Gene, Volumes I and II,The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif.(1987); Darnell et al., Molecular Cell Biology, Scientific AmericanBooks, Inc., New York, N.Y. (1986); Lewin, Genes II, John Wiley & Sons,New York, N.Y. (1985); Old, et al., Principles of Gene Manipulation: AnIntroduction to Genetic Engineering, 2d edition, University ofCalifornia Press, Berkeley, Calif. (1981); Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989); and Ausubel et al, Current Protocols in MolecularBiology, Wiley Interscience, NY, (1987, 1992). These references areherein entirely incorporated by reference as are the references citedtherein.

Assays for Plasmin Binding and Inhibition

Any suitable method may be used to test the compounds of this invention.Scatchard (Ann NY Acad Sci (1949) 51:660-669) described a classicalmethod of measuring and analyzing binding which is applicable to proteinbinding. This method requires relatively pure protein and the ability todistinguish bound protein from unbound.

A second appropriate method of measuring K_(D) is to measure theinhibitory activity against the enzyme. If the K_(D) to be measured isin the 1 nM to 1 μM range, this method requires chromogenic orfluorogenic substrates and tens of micrograms to milligrams ofrelatively pure inhibitor. For the proteins of this invention, havingK_(D) in the range 5 nM to 50 pM, nanograms to micrograms of inhibitorsuffice. When using this method, the competition between the inhibitorand the enzyme substrate can give a measured K_(i) that is higher thanthe true K_(i). Measurement reported here are not so corrected becausethe correction would be very small and the any correction would reducethe K_(i). Here, we use the measured K_(i) as a direct measure of KD.

A third method of determining the affinity of a protein for a secondmaterial is to have the protein displayed on a genetic package, such asM13, and measure the ability of the protein to adhere to the immobilized“second material”. This method is highly sensitive because the geneticpackages can be amplified. We obtain at least semiquantitative valuesfor the binding constants by use of a pH step gradient. Inhibitors ofknown affinity for the protease are used to establish standard profilesagainst which other phage-displayed inhibitors are judged. Any othersuitable method of measuring protein binding may be used.

Preferably, the proteins of this invention have a K_(D) for plasmin ofat most about 5 nM, more preferably at most about 300 pM, and mostpreferably 100 pM or less. Preferably, the binding is inhibitory so thatK_(i) is the same as K_(D). The K_(i) of QS4 for plasmin is about 2 nM.The K_(i) of SPI11for plasmin is about 88 pM.

Pharmaceutical Methods and Preparations

The preferred subject of this invention is a mammal. The invention isparticularly useful in the treatment of humans, but is suitable forveterinary applications too.

Herein, “protection” includes “prevention”, “suppression”, and“treatment”. “Prevention” involves administration of drug prior to theinduction of disease. “Suppression” involves administration of drugprior tog the clinical appearance of disease. “Treatment” involvesadministration of drug after the appearance of disease.

In human and veterinary medicine, it may not be possible to distinguishbetween “preventing” and “suppressing” since the inductive event(s) maybe unknown or latent, or the patient is not ascertained until after theoccurrence of the inductive event(s). We use the term “prophylaxis” asdistinct from “treatment” to encompass “preventing” and “suppressing”.Herein, “protection” includes “prophylaxis”. Protection need not byabsolute to be useful.

Proteins of this invention may be administered, by any means,systemically or topically, to protect a subject against a disease oradverse condition. For example, administration of such a composition maybe by any parenteral route, by bolus injection or by gradual perfusion.Alternatively, or concurrently, administration may be by the oral route.A suitable regimen comprises administration of an effective amount ofthe protein, administered as a single dose or as several doses over aperiod of hours, days, months, or years.

The suitable dosage of a protein of this invention may depend on theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the desired effect.However, the most preferred dosage can be tailored to the individualsubject, as is understood and determinable by one of skill in the art,without undue experimentation by adjustment of the dose in ways known inthe art.

For methods of preclinical and clinical testing of drugs, includingproteins, see, e.g., Berkow et al, eds., The Merck Manual, 15th edition,Merck and Co., Rahway, N.J., 1987; Goodman et al., eds., Goodman andGilman's The Pharmacological Basis of Therapeutics, 8th edition,Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery's Drug Treatment:Principles and Practice of Clinical Pharmacology and Therapeutics, 3rdedition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987),Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985), whichreferences and references cited there are hereby incorporated byreference.

In addition to a protein here disclosed, a pharmaceutical compositionmay contain pharmaceutically acceptable carriers, excipients, orauxiliaries. See, e.g., Berker, supra, Goodman, supra, Avery, supra andEbadi, supra.

In Vitro Diagnostic Methods and Reagents

Proteins of this invention may be applied in vitro to any suitablesample that might contain plasmin to measure the plasmin present. To doso, the assay must include a Signal Producing System (SPS) providing adetectable signal that depends on the amount of plasmin present. Thesignal may be detected visually or instrumentally. Possible signalsinclude production of colored, fluorescent, or luminescent products,alteration of the characteristics of absorption or emission of radiationby an assay component or product and precipitation or agglutination of acomponent or product.

The component of the SPS most intimately associated with the diagnosticreagent is called the “label”. A label may be, e.g., a radioisotope, afluorophore, an enzyme, a co-enzyme, an enzyme substrate, anelectron-dense compound, or an agglutinable particle. A radioactiveisotope can be detected by use of, for example, a γ counter or ascintillation counter or by autoradiography. Isotopes which areparticularly useful are ³H, ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C, and, preferably, ¹²⁵I.It is also possible to label a compound with a fluorescent compound.When the fluorescently labeled compound is exposed to light of theproper wave length, its presence can be detected. Among the mostcommonly used fluorescent labelling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde, and fluorescamine. Alternatively, fluorescence-emittingmetals, such as ¹²⁵Eu or other lanthanide, may be attached to thebinding protein using such metal chelating groups asdiethylenetriaminepentaacetic acid or ethylenediamine-tetraacetic acid.The proteins also can be detectably labeled by coupling to achemiluminescent compound, such as luminol, isolumino, theromaticacridinium ester, imidazole, acridinium salt, and oxalate ester.Likewise, a bioluminescent compound, such as luciferin, luciferase andaequorin, may be used to label the binding protein. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Enzyme labels, such as horseradish peroxidase and alkalinephosphatase, are preferred.

There are two basic t of assays: heterogeneous and homogeneous. Inheterogeneous assays, binding of the affinity molecule to analyte doesnot affect the label; thus, to determine the amount of analyte, boundlabel must be separated from free label. In homogeneous assays, theinteraction does affect the activity of the label, and analyte can bemeasured without separation.

In general, a plasmin-binding protein (PBP) may be used diagnosticallyin the same way that an antiplasmin antibody is used. Thus, depending onthe assay format, it may be used to assay plasmin, or, by competitiveinhibition, other substances which bind plasmin.

The sample will normally be a biological fluid, such as blood, urine,lymph, semen, milk, or cerebrospinal fluid, or a derivative thereof, ora biological tissue, e.g., a tissue section or homogenate. The samplecould be anything. If the sample is a biological fluid or tissue, it maybe taken from a human or other mammal, vertebrate or animal, or from aplant. The preferred sample is blood, or a fraction or derivativethereof.

In one embodiment, the plasmin-binding protein (PBP) is immobilized, andplasmin in the sample is allowed to compete with a known quantity of alabeled or specifically labelable plasmin analogue. The “plasminanalogue” is a molecule capable of competing with plasmin for binding tothe PBP, which includes plasmin itself. It may be labeled already, or itmay be labeled subsequently by specifically binding the label to amoiety differentiating the plasmin analogue from plasmin. The phases areseparated, and the labeled plasmin analogue in one phase is quantified.

In a “sandwich assay”, both an insolubilized plasmin-binding agent(PBA), and a labeled PBA are employed. The plasmin analyte is capturedby the insolubilized PBA and is tagged by the labeled PBA, forming atertiary complex. The reagents may be added to the sample in any order.The PBAs may be the same or different, and only one PBA need be a PBPaccording to this invention (the other may be, e.g., an antibody). Theamount of labeled PBA in the tertiary complex is directly proportionalto the amount of plasmin in the sample.

The two embodiments described above are both heterogeneous assays. Ahomogeneous assay requires only that the label be affected by thebinding of the PBP to plasmin. The plasmin analyte may act as its ownlabel if a plasmin inhibitor is used as a diagnostic reagent.

A label may be conjugated, directly or indirectly (e.g., through alabeled anti-PBP antibody), covalently (e.g., with SPDP) ornoncovalently, to the plasmin-binding protein, to produce a diagnosticreagent. Similarly, the plasmin binding protein may be conjugated to asolid phase support to form a solid phase (“capture”) diagnosticreagent. Suitable supports include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, and magnetite. The carrier canbe soluble to some extent or insoluble for the purposes of thisinvention. The support material may have any structure so long as thecoupled molecule is capable of binding plasmin.

In Vivo Diagnostic Uses

A Kunitz domain that binds very tightly to plasmin can be used for invivo imaging. Diagnostic imaging of disease foci was considered one ofthe largest commercial opportunities for monoclonal antibodies, but thisopportunity has not been achieved. Despite considerable effort, only twomonoclonal antibody-based imaging agents have been approved. Thedisappointing results obtained with monoclonal antibodies is due inlarge measure to:

-   i) Inadequate affinity and/or specificity;-   ii) Poor penetration to target sites;-   iii) Slow clearance from nontarget sites;-   iv) Immunogenicity (most are murine); and-   v) High production cost and poor stability.

These limitations have led most in the diagnostic imaging field to beginto develop peptide-based imaging agents. While potentially solving theproblems of poor penetration and slow clearance, peptide-based imagingagents are unlikely to possess adequate affinity, specificity and invivo stability to be useful in most applications.

Engineered proteins are uniquely suited to the requirements for animaging agent. In particular the extraordinary affinity and specificitythat is obtainable by engineering small, stable, human-origin proteindomains having known iii vivo clearance rates and mechanisms combine toprovide earlier, more reliable results, less toxicity/side effects,lower production and storage cost, and greater convenience of labelpreparation. Indeed, it should be possible to achieve the goal ofrealtime imaging with engineered protein imaging agents. Plasmin-bindingproteins, e.g. SPI11, may be useful for localizing sites of internalhemorrhage.

Radio-labelled binding protein may be administered to the human oranimal subject. Administration is typically by injection, e.g.,intravenous or arterial or other means of administration in a quantitysufficient to permit subsequent dynamic and/or static imaging usingsuitable radio-detecting devices. The dosage is the smallest amountcapable of providing a diagnostically effective image, and may bedetermined by means conventional in the art, using known radio-imagingagents as guides.

Typically, the imaging is carried out on the whole body of the subject,or on that portion of the body or organ relevant to the condition ordisease under study. The radio-labelled binding protein has accumulated.The amount of radio-labelled binding protein accumulated at a givenpoint in time in relevant target organs can then be quantified.

A particularly suitable radio-detecting device is a scintillationcameras, such as a γ camera. The detection device in the camera sensesand records (and optional digitizes) the radioactive decay. Digitizedinformation can be analyzed in any suitable way, many of which are knownin the art. For example, a time-activity analysis can illustrate uptakethrough clearance of the radio-labelled binding protein by the targetorgans with time.

Various factors are taken into consideration in picking an appropriateradioisotope. The isotope is picked: to allow good quality resolutionupon imaging, to be safe for diagnostic use in humans and animals, and,preferably, to have a short half-life so as to decrease the amount ofradiation received by the body. The radioisotope used should preferablybe pharmacologically inert, and the quantities administered should nothave substantial physiological effect. The binding protein may beradio-labelled with different isotopes of iodine, for example ¹²³I,¹²⁵I, or ¹³¹I (see, for example, U.S. Pat. No. 4,609,725). The amount oflabeling must be suitably monitored.

In applications to human subjects, it may be desirable to useradioisotopes other than ¹²⁵I for labelling to decrease the totaldosimetry exposure of the body and to optimize the detectability of thelabelled molecule. Considering ready clinical availability for use inhumans, preferred radio-labels include: ^(99m)Tc, ⁶⁷Ga, ⁶⁸Ga, ⁹⁰Y,¹¹¹In, ^(113m)In, ¹²³I, ¹⁸⁶Re, ¹⁸⁸Re or ²¹¹At. Radio-labelled proteinmay be prepared by various methods. These include radio-halogenation bythe chloramine-T or lactoperoxidase method and subsequent purificationby high pressure liquid chromatography, for example, see Gutkowska et alin “Endocrinology and Metabolism Clinics of America: (1987) 16 (1):183.Other methods of radio-labelling can be used, such as IODOBEADS™.

A radio-labelled protein may by administered by any means that enablesthe active agent to reach the agent's site of action in a mammal.Because proteins are subject to digestion when administered orally,parenteral administration, i.e., intravenous subcutaneous,intramuscular, would ordinarily be used to optimize absorption.

Other Uses

The plasmin-binding proteins of this invention may also be used topurify plasmin from a fluid, e.g., blood. For this purpose, the PBP ispreferably immobilized on an insoluble support. Such supports includethose already mentioned as useful in preparing solid phase diagnosticreagents.

Proteins can be used as molecular weight markers for reference in theseparation or purification of proteins. Proteins may need to bedenatured to serve as molecular weight markers. A second general utilityfor proteins is the use of hydrolyzed protein as a nutrient source.Proteins may also be used to increase the viscosity of a solution.

The protein of his invention may be used for any of the foregoingpurposes, as well as for therapeutic and diagnostic purposes asdiscussed further earlier in this specification.

Preparation of Peptides

Chemical polypeptide synthesis is a rapidly evolving area in the art,and methods of solid phase polypeptide synthesis are well-described inthe following references, hereby entirely incorporated by reference:(Merrifield, J Amer Chem Soc 85:2149-2154 (1963); Merrifield, Science232:341-347 (1986); Wade et al., Biopolymers 25:S21-S37 (1986); Fields,Int J Polypeptide Prot Res 35:161 (1990); MilliGen Report Nos. 2 and 2a,Millipore Corporation, Bedford, Mass., 1987) Ausubel et al, supra, andSambrook et al, supra. Tan and Kaiser (Biochemistry, 1977, 16:1531-41)synthesized BPTI and a homologue eighteen years ago.

As is known in the art, such methods involve blocking or protectingreactive functional groups, such as free amino, carboxyl and thiogroups. After polypeptide bond formation, the protective groups areremoved. Thus, the addition of each amino acid residue requires severalreaction steps for protecting and deprotecting. Current methods utilizesolid phase synthesis, wherein the C-terminal amino acid is covalentlylinked to an insoluble resin particles that can be filtered. Reactantsare removed by washing the resin particles with appropriate solventsusing an automated machine. Various methods, including the “tBoc” methodand the “Fmoc” method are well known in the art. See, inter alia,Atherton et al., J Chem Soc Perkin Trails 1:538-546 (1981) and Sheppardet al., Int J Polypeptide Prot Res 20:451-454 (1982).

EXAMPLES Example 1 Construction of LACI (K1) Library

A synthetic oligonucleotide duplex having NsiI- and MluI-compatible endswas cloned into a parental vector (LACI-K1::IU) previously cleaved withthe above two enzymes. The resultant ligated material was transfected byelectroporation into XLIMR (F⁻) E. coli strain and plated on ampicillin(Ap) plates to obtain phage-generating Ap^(R) colonies. The variegationscheme for Phase 1 focuses on the P1 region, and affected residues 13,16, 17, 18 and 19. It allowed for 6.6×10⁵ different DNA sequences(3.1×10⁵ different protein sequences). The library obtained consisted of1.4×10⁶ independent cfu's which is approximately a two foldrepresentation of the whole library. The phage stock generated from thisplating gave a total titer of 1.4×10¹³ pfu's in about 3.9 ml, with eachindependent done being represented, on average, 1×10⁷ in total and2.6×10⁶ times per ml of phage stock.

To allow for variegation of residues 31, 32, 34 and 39 (phase II),synthetic oligonucleotide duplexes with MluI- and BstEII-compatible endswere cloned into previously cleaved R_(f) DNA derived from one of thefollowing

-   i) the parental construction,-   ii) the phase I library, or-   iii) display phage selected from the first phase binding to a given    target.

The variegation scheme for phase II allows for 4096 different DNAsequences (1600 different protein sequences) due to alterations atresidues 31, 32, 34 and 39. The final phase II variegation is dependentupon the level of variegation remaining following the three rounds ofbinding and elution with a given target in phase I.

The combined possible variegation for both phases equals 2.7×10⁸different DNA sequences or 5.0×10⁷ different protein sequences. Whenpreviously selected display phage are used as the origin of R_(f) DNAfor the phase II variegation, the final level of variegation is probablyin the range of 10 ⁵ to 10⁶.

Example 2 Screening of LACI-K1 Library for Binding to Plasmin

The scheme for selecting LACI-K1 variants that bind plasmin involvesincubation of the phage-display library with plasmin-beads (Calbiochem,San Diego, Calif.; catalogue no. 527802) in a buffer (PBS containing 1mg/ml BSA) before washing away unbound and poorly retained display-phagevariants with PBS containing 0.1% Tween 20. The more strongly bounddisplay-phage are eluted with a low pH elution buffer, typically citratebuffer (pH 2.0) containing 1 mg/ml BSA, which is immediately neutralizedwith Tris buffer to pH 7.5. This process constitutes a single round ofselection.

The neutralized eluted display-phage can be either used:

-   i) to inoculate an F⁺ strain of E. coli to generate a new    display-phage stock, to be used for subsequent rounds of selection    (so-called conventional screening), or-   ii) be used directly for another immediate round of selection with    the protease beads (so-called quick screening).

Typically, three rounds of either method, or a combination of the two,are performed to give rise to the final selected display-phage fromwhich a representative number are sequenced and analyzed for bindingproperties either as pools of display-phage or as individual clones.

For the LACI-K1 library, two phases of selection were performed, eachconsisting of three rounds of binding and elution. Phase I selectionused the phase I library (variegated residues 13, 16, 17, 18, and 19)which went through three rounds of binding and elution against plasmingiving rise to a subpopulation of clones. The R_(f) DNA derived fromthis selected subpopulation was used to generate the Phase II library(addition of variegated residues 31, 32, 34 and 39). About 5.6×10⁷independent transformants were obtained. The phase II librariesunderwent three further rounds of binding and elution with the sametarget protease giving rise to the final selectants.

Following two phases of selection against plasmin-agarose beads arepresentative number (16) of final selection display-phage weresequenced. Table 2 shows the sequences of the selected LACI-K1 domainswith the amino acids selected at the variegated positions in upper case.Note the absolute selection of residues P₁₃, A₁₆, R₁₇, F₁₁, and E₁₉.There is very strong selection for E at 31 and Q at 32. There is noconsensus at 34; the observed amino acids are {T₃, Y₂, H₂, D, R, A, V₂,I₃, and L}. The amino acids having side groups that branch at C₀ (T, I,and V) are multiply represented and are preferred. At position 39, thereis no strong consensus (G₆, D₃, Q₂, A₂, R, F, E), but G, D, Q, and Aseem to be preferred (in that order).

A separate screening of the LACI-K1 library against plasmin gave a verysimilar consensus from 16 sequenced selected display-phage. Thesesequences are shown in Table 3 (selected residues in upper case). Thesesequences depart from those of Table 2 in that E here predominates atposition 19. There is a consensus at 34 (T₃, V₃, S₃, I₂, L, A, F) of T,V, or S. Combining the two sets, there is a preference for (in order ofpreference) T, V, I, S, A, H, Y, and L, with F, D, and R being allowed.

Expression, Purification and Kinetic Analysis.

The three isolates QS4, ARFK#1, and ARFK#2 were recloned into a yeastexpression vector. The yeast expression vector is derived from pMFalpha8(KURJ82 and MIYA85). The LACI variant genes were fused to part of thematα 1 gene, generating a hybrid gene consisting of the matα 1promoter-signal peptide,and leader sequence-fused to the LACI variant.The cloning site is shown in Table 24. Note that the correctly processedLACI-K1 variant protein should be as detailed in Table 2 and Table 3with the addition of residues glu-ala-ala-glu to the N-terminal met(residue 1 in Table 2 and Table 3). Expression in S. cerevisiae gave ayield of about 500 μg of protease inhibitor per liter of medium.Yeast-expressed LACI (kunitz domain 1), BPTI and LACI variants: QS4,ARFK#1 and ARFK#2 were purified by affinity chromatography usingtrypsin-agarose beads.

The most preferred production host is Pichia pastoris utilizing thealcohol oxidase system Others have produced a number of proteins in theyeast Pichia pastoris. For example, Vedvick et al. (VEDV91) and Wagneret al. (WAGN92) produced aprotinin from the alcohol oxidase promoterwith induction by methanol as a secreted protein in the culture mediumat ≈1 mg/ml. Gregg et al. (GREG93) have reviewed production of a numberof proteins in P. pastoris. Table 1 of GREG93 shows proteins that havebeen produced in P. pastoris and the yields.

Kinetic Data

Inhibition of hydrolysis of succinyl-Ala-Phe-Lys-(F₃Ac)AMC (a methylcoumarin) (Sigma Chemical, St. Louis, Mo.) by plasmin at 2.5×10⁻¹ M withvarying amount of inhibitor were fit to the standard form for atight-binding substrate by least-squares. Preliminary kinetic analysisof the two ARFK variants demonstrated very similar inhibitory activityto that of the QS4 variant.) These measurements were carried out withphysiological amounts of salt (150 mM) so that the affinities arerelevant to the action of the proteins in blood.

Table 23 shows that QS4 is a highly specific inhibitor of human plasmin.Phage that display the LACI-K1 derivative QS4 bind to plasmin beads atleast 50-times more than it binds to other protease targets.

New Library for Plasmin:

A new library of LACI-K1 domains, displayed on M13 gIIIp and containingthe diversity shown in Table 5 was made and screened for plasminbinding. Table 6 shows the sequences selected and the consensus. Wecharacterized the binding of the selected proteins by comparing thebinding of clonally pure phage to BPTI display phage. Isolates 11, 15,08, 23, and 22 were superior to BPTI phage. We produced solubleSPI11(Selected Plasmin Inhibitor #11) and tested its inhibitoryactivity, obtaining a K_(i) of 88 pM which is at least two-fold betterthan BPTI. Thus, we believe that the selectants SPI15, SPI08, and SPI22are far superior to BPTI and that SPI23 is likely to be about as potentas BPTI. All of the listed proteins are much closer to a human proteinamino-acid sequence than is BPTI and so have less potential forimmunogenicity.

All references, including those to U.S. and foreign patents or patentapplications, and to nonpatent disclosures, are hereby incorporated byreference in their entirety.

TABLE 1 Sequence of whole LACI: (SEQ ID NO. 1)   5          5          5          5 1 MIYTHKKVHA LWASVCLLLN LAPAPLNAdseedeehtiit    5 dtelpplklM 51 HSFCAFKADD GPCKAIMKRF FFNIFTRQCEEFIYGGCEGN QNRFESLEEC 101 KKMCTRDnan riikttlqqe kpdfCfleed pgicrgyitryfynnqtkqC 151 erfkyggClg nmnnfetlee CkniCedg pn gfqvdnyqtq lnavnnsltp201 qstkvpslfe fhgpswCltp adrglCrane nrfyynsvig kcrpfkysgC 251ggnennftsk qeClraCkkg fiqriskggl iktkrkrkkq rvkiayeeif 301 vknm Thesignal sequence (1-28) is uppercase and underscored LACI-K1 is uppercaseLACI-K2 is underscored LACI-K3 is bold

TABLE 2 Sequence of LACI-K1 and derivatives that bind human plasmin         1         2         3         4         51234567890123456789012345678901234567890123456789012345678 LACI-K1mhsfcafkaddgpckaimkrfffniftrqceefiyggcegnqnrfesleeckkmctrd SEQ ID NO. 2QS1 mhsfcafkaddgPckARFErfffniftrqcEQfTyggcRgnqnrfesleeckkmctrd SEQ IDNO. 3 QS4 mhsfcafkaddgPckARFErfffniftrqcEQfYyggcDgnqnrfesleeckkmctrd SEQID NO. 4 QS7 mhsfcafkaddgPckARFErfffniftrqcEQfHyggcDgnqnrfesleeckkmctrdSEQ ID NO. 5 QS8mhsfcafkaddgPckARFErfffniftrqcEQfDyggcAgnqnrfesleeckkmctrd SEQ ID NO. 6QS9 mhsfcafkaddgPckARFErfffniftrqcQEfRyggcDgnqnrfesleeckkmctrd SEQ IDNO. 7 QS13 mhsfcafkaddgPckARFErfffniftrqcQQfYyggcQgnqnrfesleeckkmctrdSEQ ID NO. 8 QS15mhsfcafkaddgPckARFErfffniftrqcEEfAyggcGgnqnrfesleeckkmctrd SEQ ID NO. 9NS2 mhsfcafkaddgPckARFErfffniftrqcQQfVyggcGgnqnrfesleeckkmctrd SEQ IDNO. 10 NS4 mhsfcafkaddgPckARFErfffniftrqcEQfTyggcGgnqnrfesleeckkmctrdSEQ ID NO. 11 NS6mhsfcafkaddgPckARFErfffniftrqcEEfTyggcGgnqnrfesleeckkmctrd SEQ ID NO. 12NS9 mhsfcafkaddgPckARFErfffniftrqcEQfIyggcQgnqnrfesleeckkmctrd SEQ IDNO. 13 NS11 mhsfcafkaddgPckARFErfffniftrqcEQfIyggcGgnqnrfesleeckkmctrdSEQ ID NO. 14 NS12mhsfcafkaddgPckARFErfffniftrqcEQfIyggcFgnqnrfesleeckkmctrd SEQ ID NO. 15NS14 mhsfcafkaddgPckARFErfffniftrqcQQfHyggcEgnqnrfesleeckkmctrd SEQ IDNO. 16 NS15 mhsfcafkaddgPckARFErfffniftrqcEQfVyggcAgnqnrfesleeckkmctrdSEQ ID NO. 17 NS16mhsfcafkaddgPckARFErfffniftrqcEQfLyggcGgnqnrfesleeckkmctrd SEQ ID NO. 18ARFRCON mhsfcafkaddgPckARFErfffniftrqcEQfiyggcGgnqnrfesleeckkmctrd SEQID NO. 19

TABLE 3 Sequence of LACI-K1 and derivatives that bind human plasmin         1         2         3         4         51234567890123456789012345678901234567890123456789012345678 LACI-K1mhsfcafkaddgpckaimkrfffniftrqceefiyggcegnqnrfesleeckkmctrd SEQ ID NO. 2ARFK#1 mhsfcafkaddgPckARFErfffniftrqcEQfVyggcGgnqnrfesleeckkmctrd SEQ IDNO. 20 ARFK#2 mhsfcafkaddgPckARFErfffniftrqcEEfVyggcGgnqnrfesleeckkmctrdSEQ ID NO. 21 ARFK#3mhsfcafkaddgLckGRFQrfffniftrqcEEfIyggcEgnqnrfesleeckkmctrd SEQ ID NO. 22ARFK#4 mhsfcafkaddgPckARFErfffniftrqcEQfTyggcMgnqnrfesleeckkmctrd SEQ IDNO. 23 ARFK#5 mhsfcafkaddgPckARFErfffniftrqcEQfSyggcGgnqnrfesleeckkmctrdSEQ ID NO. 24 ARFK#6mhsfcafkaddgPckARFErfffniftrqcEEfLyggcLgnqnrfesleeckkmctrd SEQ ID NO. 25ARFK#7 mhsfcafkaddgPckARFErfffniftrqcEQfSyggcQgnqnrfesleeckkmctrd SEQ IDNO. 26 ARFK#8 mhsfcafkaddgPckARFErfffniftrqcEQfAyggcAgnqnrfesleeckkmctrdSEQ ID NO. 27 ARFK#9mhsfcafkaddgPckARFErfffniftrqcEQfIyggcVgnqnrfesleeckkmctrd SEQ ID NO. 28ARFK#10 mhsfcafkaddgPckARFErfffniftrqcEEfSyggcKgnqnrfesleeckkmctrd SEQID NO. 29 ARFK#11mhsfcafkaddgPckARFErfffniftrqcEEfVyggcKgnqnrfesleeckkmctrd SEQ ID NO. 30ARFK#12 mhsfcafkaddgPckASFErfffniftrqcEQfTyggcNgnqnrfesleeckkmctrd SEQID NO. 31 ARFK#13mhsfcafkaddgPckASFErfffniftrqcEEfTyggcLgnqnrfesleeckkmctrd SEQ ID NO. 32ARFK#14 mhsfcafkaddgPckARFErfffniftrqcEQfFyggcHgnqnrfesleeckkmctrd SEQID NO. 33 ARFK#15mhsfcafkaddgPckARFErfffniftrqcEQfTyggcGgnqnrfesleeckkmctrd SEQ ID NO. 34ARFK#16 mhsfcafkaddgPckARFErfffniftrqcEQfTyggcMgnqnrfesleeckkmctrd SEQID NO. 35 ARFKC01mhsfcafkaddgPckARFErfffniftrqcEQfTyggcGgnqnrfesleeckkmctrd SEQ ID NO. 36ARFKC02 mhsfcafkaddgPckARFErfffniftrqcEQfVyggcGgnqnrfesleeckkmctrd SEQID NO. 37

TABLE 4 Kunitz domains, some of which inhibit plasmin Amino-acidSequence Affin- Protein         111111111122222222223333333333444444444A555555555 ityidentifier 1234567890123456789012345678901234567890123456789012345678K_(D) SEQ ID NO. QS4mhsfcafkaddgPckARFErfffniftrqcEQfYyggcDgnqnrfesleeckkmctrd 2 nM SEQ IDNO. 4 NS4 mhsfcafkaddgPckARFErfffniftrqcEQfTyggcGgnqnrfesleeckkmctrd (B)SEQ ID NO. 11 BPTIRPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA .3 nM SEQ IDNO. 38 Human VREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSA75 pM SEQ ID NO. 39 APP-I (KIDO88) 225 nM (DENN94a) SpI11mhsfcafkaETgPcRARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrd 88 pM SEQ IDNO. 40 SpI15 mhsfcafkaESgPcRARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrd(A) SEQ ID NO. 41 SPI08mhsfcafkaDGgPcRARFErFffniftrqceefiyggcegnqnrfesleeckkmctrd (A) SEQ IDNO. 42 SPI23 mhsfcafkaEGgPcRAKFQrWffniftrqceefiyggcegnqnrfesleeckkmctrd~.5 nM SEQ ID NO. 43 SPI22mhsfcafkaDGgPcKGKFPrFffniftrqceefiyggcegnqnrfesleeckkmctrd >2 nM SEQ IDNO. 44 SPIcon1mhsfcafkaETgPcRAkFDrWffniftrqcEAfVyggcGgnqnrfesleeckkmctrd SEQ ID NO. 45SPI60 mhsfcafkaETgPcRAkFDrWffniftrqcEPfVYggcEgnqnrfesleeckkmctrd (B) SEQID NO. 46 SPI59mhsfcafkaETgPcRAkFDrWffniftrqcNTfVYggcGgnqnrfesleeckkmctrd SEQ ID NO. 47SPI42 mhsfcafkaETgPcRGkFDrWffniftrqcQGfVYggcGgnqnrfesleeckkmctrd SEQ IDNO. 48 SPI55 mhsfcafkaEVgPcRAkFDrWffniftrqcHLfTYggcGgnqnrfesleeckkmctrdSEQ ID NO. 49 SPI56mhsfcafkaETgPcRGkFDrWffniftrqcAQfVYggcEgnqnrfesleeckkmctrd SEQ ID NO. 50SPI43 mhsfcafkaETgPcRGkFDrWffniftrqcESfHYggcKgnqnrfesleeckkmctrd >−4 nMSEQ ID NO. 51 SPI52mhsfcafkaDAgPcRAkFErFffniftrqcEAfLYggcGgnqnrfesleeckkmctrd SEQ ID NO. 52SPI46 mhsfcafkaDVgPcRAkFErFffniftrqcEAfLYggcEgnqnrfesleeckkmctrd SEQ IDNO. 53 SPI51 mhsfcafkaDAgPcRAkFErFffniftrqcTAfFYggcGgnqnrfesleeckkmctrd~.5 nM SEQ ID NO. 54 SPI54mhsfcafkaDSgPcRARFDrWffniftrqcTRfPYggcGgnqnrfesleeckkmctrd >−4 nM SEQ IDNO. 55 SPI49 mhsfcafkaETgPcRAkIPrLffniftrqcEPfIWggcGgnqnrfesleeckkmctrdSEQ ID NO. 56 SPI47mhsfcafkaDAgPcRAkFErFffniftrqcEEfIYggcEgnqnrfesleeckkmctrd −.8 nM SEQ IDNO. 57 SPI53 mhsfcafkaETgPcKGSFDrWffniftrqcNVfRYggcRgnqnrfesleeckkmctrdSEQ ID NO. 58 SPI41mhsfcafkaDAgPcRARFErFffniftrqcDTfLYggcEgnqnrfesleeckkmctrd (AB) SEQ IDNO. 59 SPI57 mhsfcafkaDSgPcKGRFGrLffniftrqcTAfDWggcGgnqnrfesleeckkmctrdSEQ ID NO. 60 DPI-1.1.1mhsfcafkadTgpcRaRFDrfffniftrqceAfiyggcegnqnrfesleeckkmctrd (A) SEQ IDNO. 61 DPI-1.1.2mhsfcafkadTgpcRaRFDrfffniftrqceefiyggcegnqnrfesleeckkmctrd (A) SEQ IDNO. 62 DPI-1.1.3mhsfcafkadAgpcRaRFDrfffniftrqceefiyggcegnqnrfesleeckkmctrd (A) SEQ IDNO. 63 DPI-1.1.4mhsfcafkadTgpckaRFDrfffniftrqceAfiyggcegnqnrfesleeckkmctrd (AB) SEQ IDNO. 127 DPI-1.1.5mhsfcafkaddgpckaRFDrfffniftrqceefiyggcegnqnrfesleeckkmctrd (B) SEQ IDNO. 128 DPI-1.1.6mhsfcafkaddgpckaRFkrfffniftrqceefiyggcegnqnrfesleeckkmctrd (C) SEQ IDNO. 129 Human KPDFCFLEEDPGICRGYITRYFYNNQTKQCERFKYGGCLGNMNNFETLEECKNICEDGSEQ ID NO. 64 LACI-K2 DPI-1.2.1kpdfcfleedTgPcrgRFDryfynnqtkqceTfIyggcEgnmnnfetleecknicedg SEQ ID NO. 65Human GPSWCLTPADRGLCRANENRFYYNSVIGKCRPFKYSGCGGNENNFTSKQECLRACKKG SEQ IDNO. 66 LACI-K3 DPI-1.3.1gpswcltpadTgPcraRFDrfyynsvigkcEpfIyGgcggnennftskqeclrackkg SEQ ID NO. 67Human ETDICKLPKDEGTCRDFILKWYYDPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV SEQ IDNO. 68 collagen α3 KuDom DPI-2.1etdicklpkdTgPcrARFDkwyydpntkscEPfVyggcggnenkfgsqkecekvcapv SEQ ID NO. 69Human NAEICLLPLDYGPCRALLLRYYYDRYTQSCRQFLYGGCEGNANNFYTWEACDDACWRI SEQ IDNO. 70 TFPI-2 DOMAIN 1 DPI-3.1.1naeicllpldTgpcraRFDryyydrytqscEqflyggcegnannfytweacddacwri SEQ ID NO. 71Human VPKVCRLQV- SEQ ID NO. 72 TFPI-2        SVDDQCEGSTEKYFFNLSSMTCEKFFSGGCHRNR- DOMAIN 2                                        IENRFPDEATCMGFCAPK DPI-3.2.1vpkvcrlqvETGPcRgKFekyffnlssmtceTfvYggcEGnrnrfpdeatcmgfcapk SEQ ID NO. 73Human IPSFCYSPKDEGLCSANVTRYYFNPRYRTCDAFTYTGCGGNDNNFVSREDCKRACAKA SEQ IDNO. 74 TFPI-2 DOMAIN 3 DP1-3.3.1ipsfcyspkdTgPcRaRFtryyfnpryrtcdaftyGgcggndnnfvsredckracaka SEQ ID NO. 75HUMAN KEDSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTCRTV SEQ IDNO. 76 ITI-K1 DPI-4.1.1kedscqlgyEagpcRgKFsryfyngtsmacetfVyggcGgngnnfvtekeclqtcrtv SEQ ID NO. 77Human TVAACNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCGVP SEQ IDNO. 78 ITI-K2 DPI-4.2.1tvaacnlpiDTgpcraRFqlwafdavkgkcvlfVyggcqgngnkfysekecreycgvp SEQ ID NO. 79PROTEASE VREVCSEQAETGPCRAMISRWYFDVTEGKCAPFFYGGCGGNRNNFDTEEYCMAVCGSA SEQID NO. 80 NEXIN-II DPI-5.1vrevcseqaetgpcraRFsrwyfdvtegkcapffyggcggnrnnfdteeycmavcgsa SEQ ID NO. 81DPI-5.2 vrevcseqaetgpcraRFsrwyfdvtegkcEpfIyggcggnrnnfdteeycmavcgsa SEQID NO. 82 HKI B9LPNVCAFPMEKGPCQTYMTRWFFNFETGECELFAYGGCGGNSNNFLRKEKCEKFCKFT SEQ ID NO.124 DPI-6.1 lpnvcafpmeTgpcRARFtrwffnfetgecelfayggcggnsnnflrkekcekfckftSEQ ID NO. 125 DPI-6.2lpnvcafpmeTgpcRARFDrwffnfetgecelfVyggcggnsnnflrkekcekfckft SEQ ID NO.126 DPI-4.2.2 tvaacnlpivTgpcraRFqlwafdavkgkcvlfpyggcqgngnkfysekecreycgvpSEQ ID NO. 130 DPI-4.2.3tvaacnlpivTgpcraRFqRwafdavkgkcvlfpyggcqgngnkfysekecreycgvp SEQ ID NO.131 DPI-4.2.4 tvaacnlpivTgpcraRFqRwafdavkgkcvlfVyggcqgngnkfysekecreycgvpSEQ ID NO. 132 DPI-4.2.5tvaacnlpiETgpcraRFDRwafdavkgkcETfVyggcGgngnkfysekecreycgvp SEQ ID NO.133 DPI-7.1 rpdfcleppytgpckarFiryfynakaglcqtfvyggcrakrnnfksaedcmrtcggaSEQ ID NO. 134 DPI-7.2rpdfcleppytgpcRarFiryfynakaglcqtfvyggcrakrnnfksaedcmrtcgga SEQ ID NO.135 DPI-7.3 rpdfcleppytgpcRarFiryfynakaglcqtfvyggcGakrnnfksaedcmrtcggaSEQ ID NO. 136 DPI-7.4                                                 ⊙SEQ ID NO. 137rpdfcleppDtgpcRarFDryfynakaglcEtfvyggcGakrnnfksaedcmrtcgga Under“Affinity”, “(A)” means the K_(D) is likely to be less than that of BPTI(viz.300 pM), “(B)” means K_(D) is likely to be less than 2 nM, and“(C)” means that K_(D) is likely to be less than 20 nM.

TABLE 5 vgDNA for LACI-DI to vary residues 10, 11, 13, 15, 16, 17, & 19for plasmin in view of App-I (now known not to be very potent)

DNA: 262,144 * 4 = 1,048,576 protein: 143,360 * 4 = 573,440 The aminoacid seq has SEQ ID NO. 85. Thes variegation allows the AppI sequence toappear in the P6-P6′ positions.

TABLE 6 LACI-K1 derivatives selected for Plasmin binding 111111111122Phage K_(D) Ident 012345678901 DIFFS Binding (pM) SEQ ID NO. ConsensusETGPCRARFERW 0 SEQ ID NO. 88 LACI-K1 ddgpckaimkrf 7 SEQ ID NO. 2 SPI31---------G-- 1 SEQ ID NO. 89 SPI11 ---------D-- 1 3.2 × 88 SEQ ID NO. 40SPI15 -S-------D-- 2 2.5 × SEQ ID NO. 90 SPI24 D-----G----L 3 SEQ ID NO.91 SPI33 ---S--G--D-- 3 SEQ ID NO. 92 SPI34 -V-----S-P-- 3 SEQ ID NO. 93SPI26 -------T-P-F 3 SEQ ID NO. 94 SPI37 -V-----S-H-- 3 SEQ ID NO. 95SPI32 D------S-G-- 3 SEQ ID NO. 96 SPI12 ------GM-P-- 3 SEQ ID NO. 97SPI36 -G-------N-F 3 SEQ ID NO. 98 SPI08 DG---------F 3 2.6 × SEQ ID NO.42 SPI38 --------IS-F 3 SEQ ID NO. 99 SPI18 -G-----K---F 3 SEQ ID NO.100 SPI23 -G-----K-Q-- 3 1.25 × SEQ ID NO. 43 SPI35 DS-A--G----- 4 SEQID NO. 101 SPI02 DS----G----F 4 0.83 × SEQ ID NO. 102 SPI25 D------S-P-L4 SEQ ID NO. 103 SPI17 -V------IQ-F 4 0.09 × SEQ ID NO. 104 SPI05-S-----K-A-F 4 0.64 × SEQ ID NO. 105 SPI13 -G-----K-A-F 4 SEQ ID NO. 106SPI07 D--S---KI--- 4 SEQ ID NO. 107 SPI03 DS---K---D-- 4 0.48 × SEQ IDNO. 108 SPI06 DG---KG----- 4 SEQ ID NO. 109 SPI16 -V-A-KG--H-- 5 0.22 ×SEQ ID NO. 110 SPI04 DG-----S-P-F 5 SEQ ID NO. 111 SPI01 DS-A---M-H-F 60.25 × SEQ ID NO. 112 SPI14 DS-A---K-R-- 5 SEQ ID NO. 113 SPI28DS-T-K---P-F 6 SEQ ID NO. 114 SPI27 -----KGKIA-F 6 SEQ ID NO. 115 SPI21DS-A-KGK---- 6 0.38 × SEQ ID NO. 116 SPI22 DG---KGK-P-F 7 2.0 × SEQ IDNO. 44 “Diffs” is the number of differences from the Consensus. “PhageBinding” is the binding of phage that display the named protein relativeto binding of phage that display BPTI.

TABLE 7 Variation of Residues 31, 32, 34, and 39

gtcgtgctctttagcacgacctg-3′ (SEQ ID NO. 86) The amino acid sequence hasSEQ ID NO. 87. The EcoRI site is erased; thus, cleavage with EcoRI canbe used to eliminate (or at least greatly reduce) parental DNA. Thereare 262,144 DNA sequences and 72,000 protein sequences.

TABLE 8 Selectants for plasmin binding with variegation of second loop111111111122 3333333334 # Diffs Id 012345678901 1234567890 C1 C K1 Con1ETgPcRAKFDrW EAfVYggcGg 10 SEQ ID NO. 45 SPI47 DA-------E-F -E-I----E- 7(5) 5 SEQ ID NO. 57 SPI51 DA-------E-F T--F------ 6 (4) 9 SEQ ID NO. 54SPI52 DA-------E-F ---L------ 5 (3) 8 SEQ ID NO. 52 SPI46 DV-------E-F---L----E- 6 (3) 7 SEQ ID NO. 53 SPI41 DA-----R-E-F DT-L----E- 9 (6) 8SEQ ID NO. 59 SPI42 ------G----- QG-------- 3 (3) 12 SEQ ID NO. 48 SPI43------G----- -S-H----K- 4 (4) 11 SEQ ID NO. 51 SPI56 ------G-----AQ------E- 4 (3) 11 SEQ ID NO. 50 SPI59 ------------ NT-------- 2 (2) 11SEQ ID NO. 47 SPI60 ------------ -P------E- 2 (1) 9 SEQ ID NO. 46 SPI55-V---------- HL-T------ 4 (4) 11 SEQ ID NO. 49 SPI49 --------IP-L-P-IW----- 6 (6) 10 SEQ ID NO. 56 SPI57 DS---KGR-G-L T--DW----- 10 (8)11 SEQ ID NO. 60 SPI53 -----KGS---- NV-R----R- 7 (7) 11 SEQ ID NO. 58SPI54 DS-----R---- TR-P------ 6 (4) 10 SEQ ID NO. 55 SPI11 -------R----eefiyggceg (1) (4) 7 SEQ ID NO. 40 LACI1 ddgpckaimkrf eefiyggceg 10 (7)0 SEQ ID NO. 2 See notes below. In the Table, “-” means that the proteinhas the consensus (Con1) type. Con1 contains the most common type ateach position; amino acids shown in Con1 were not varied. Four positions(10, 31, 34, and 39) showed significant toleration for a second type,leading to 15 subsidiary consensus sequences: Con2-Con16. The column“# Diffs” shows the number of differences from CON1 under “C1”, thedifferences with the closest of Con1-Con16 under “C”, and thedifferences from LACI-K1 under “K1”. SPI11 was selected from a libraryin which residues 31-39 were locked at the wild-type. SPI11 <BPTI <SPI23≈ SPI51 <SPI47 <QS4 <SPI22 <SPI54 <SPI43 Highly very potent Superiorpotent

TABLE 9 Conservative and Semiconservative substitutions InitialConservative Semi-conservative AA type Category substitutionsubstitution A Small non- G, S, T N, V, P, (C) polar or slightly polar Cfree SH A, M, L, V, I F, G disulfide nothing nothing D acidic, E, N, S,T, Q K, R, H, A hydrophilic E acidic, D, Q, S, T, N K, R, H, Ahydrophilic F aromatic W, Y, H, L, M I, V, (C) G Gly-only nothingnothing conformation “normal” A, S, N, T D, E, H, I, K, L, M,conformation Q, R, V H amphoteric Y, F, K, ^(o)R L, M, A, (C) aromatic Ialiphatic, V, L, M, A F, Y, W, G (C) branched β carbon K basic R, H Q,N, S, T, D, E, A L aliphatic M, I, V, A F, Y, W, H, (C) M hydrophobic L,I, V, A Q, F, Y, W, (C), (R), (K), (E) N non-polar S, T, (D), Q, K, Rhydrophilic A, G, (E) P inflexible V, I A, (C), (D), (E), F, H, (K), L,H, N, Q, (R), S, T, W, Y Q aliphatic N, E, A, S, T, D M, L, K, R plusamide R basic K, Q, H S, T, E, D, A, S hydrophilic A, T, G, N D, E, R, KT hydrophilic A, S, G, N, V D, E, R, K, I V aliphatic, I, L, M, A, T P,(C) branched β carbon W aromatic F, Y, H L, M, I, V, (C) Y aromatic F,W, H L, M, I, V, (C)

Changing from A, F, H, I, L, M, P, V, W, or Y to C is semiconservativeif the new cysteine remains as a free thiol.

Changing from M to E, R, K is semiconservative if the ionic tip of thenew side group can reach the protein surface while the methylene groupsmake hydrophobic contacts.

Changing from P to one of K, R, E, or D is semiconservative if the sidegroup is on or near the surface of the protein.

TABLE 10 Plasmin-inhibiting Kunitz domain derivatives of LACI-K1Consensus #1 Consensus #2 Consensus #3 Consensus #4 Position Type StatusType Status Type Status Type Status 10 D fixed D fixed E/D S-S D/E S-S11 D fixed D fixed T/S G-S T/A G-S 12 G fixed G fixed G fixed G fixed 13P Abs-S P VS-S P VS-S P Abs-S 14 C fixed C fixed C fixed C fixed 15 Kfixed K fixed R S-S R S-S 16 A Abs-S A Abs-S A VS-S A S-S 17 R Abs-S RVS-S R/K S-S K S-S 18 F Abs-S F Abs-S F VS-S F VS-S 19 E Abs-S E Abs-SE/P/D S-S D/E VS-S 20 R fixed R fixed R fixed R fixed 21 F fixed F fixedW/F weak- W/F weak- Sel Sel 31 E S-S E S-S E fixed E/t G-S 32 Q G-S QG-S E fixed A/T Strong for no charge, weak for type 33 F fixed F fixed Ffixed F fixed 34 — no T/S weak I fixed V/L/I Weak consensus 35 Y fixed Yfixed Y fixed Y S-S 39 — no G weak E fixed G/E some- consensus Sel. Sel.Abs-S Absolute Selection VS-S Very Strong Selection S-S Strong SelectionG-S Good Selection

TABLE 11 High Specificity Designed Plasmin Inhibitors Sequence         1111111111222222222233333333334444444444555555555 Ident1234567890123456789012345678901234567890123456789012345678 SEQ ID NO.SPI11 mhsfcafkaETgPcRARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrd SEQ IDNO. 40 SPI11-R15AmhsfcafkaETgPcAARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrd SEQ ID NO.117 SPI1-R15G mhsfcafkaETgPcGARFDrWffniftrqceefiyggcegnqnrfesleeckkmctrdSEQ ID NO. 118 SPI11-R15N-mhsfcafkaETgPcNARFDrWffniftrqceAfiyggcegnqnrfesleeckkmctrd SEQ ID NO.117 E32A

TABLE 12 vgDNA for LACI-D1 to vary residues 10, 11, 12, 13, 14, 15, 16,17, 19, 20, 21, 37, 38, and 39 for plasmin in view of App-I and SPI11

First (top) strand of DNA has SEQ ID NO. 120. Second (bottom) strand ofDNA has SEQ ID NO. 121. The amino-acid sequence has SEQ ID NO. 122. Thetop strand for codons 31-42 (shown stricken) need not be synthesized,but is produced by PCR from the strands shown. There are 1.37 × 10¹¹ DNAsequences that encode 4.66 × 10¹⁰ amino-acid sequences.

TABLE 14 Definition of a Kunitz Domain (SEQ ID NO. 123)         1         2         3         4         51234567890123456789012345678901234567890123456789012345678xxxxCxxxxxxGxCxxxxxxXXXxxxxxxCxxFxXXGCxXxxXxXxxxxxCxxxCxxx Where: X1,X2, X3, X4, X58, X57, and X56 may be absent, X21 = Phe, Tyr, Trp, X22= Tyr or Phe, X23 = Tyr or Phe, X35 = Tyr or Trp, X36 = Gly or Ser, X40= Gly or Ala, X43 = Asn or Gly, and X45 = Phe or Tyr

TABLE 15 Substitutions to confer high affinity for plasmin on KuDomsPosition Allowed types 10 Asp, Glu, Tyr 11 Thr, Ala, Ser, Val, Asp 12Gly 13 Pro, Leu, Ala 14 Cys 15 Arg, Lys 16 Ala, Gly 17 Arg, Lys, Ser 18Phe, Ile 19 Glu, Asp, Pro, Gly, Ser, Ile 20 Arg 21 Phe, Trp, Tyr 31 Asp,Glu, Thr, Val, Gln, Ala 32 Thr, Ala, Glu, Pro, Gln 34 Val, Ile, Thr,Leu, Phe, Tyr, His, Asp, Ala, Ser 35 Tyr, Trp 36 Gly 37 Gly 38 Cys 39Glu, Gly, Asp, Arg, Ala, Gln, Leu, Lys, MetIn Table 15 the bold reside types are preferred

TABLE 16 Summary of Sequences selected from First LACI-K1 library forbinding to Plasmin BPTI # Residues Allowed (BPTI type) (LACI-K1) inLibrary Preferred Residues 13 (P) P LHPR PL 16 (A) A AG AG 17 (R) IFYLHINA RS SCPRTVDG 18 (I) M all F 19 (I) K LWQMKAG EQ SPRTVE 31 (Q) EEQ EQ 32 (T) E EQ QE 34 (V) I all TYHDRAVILSF 39 (R) E all GADRQFEMLVKNH

TABLE 17 Distribution of sequences selected from first library: PositionA C D E F G H I K L M N P Q R S T V W Y 13 x x x x x x 0 x x 1 x x 31* x0 x x x x x 16 31* x x x x 1 x x x x x x x x x x x x x x 17 0 0 0 x 0 00  0* x 0 x 0 0 x 30  2 0 0 x 0 18 0 0 0 0 32  0 0 0 0 0  0* 0 0 0 0 0 00 0 0 19 0 x x 31  x 0 x x  0* 0 0 x 0 1 0 0 0 0 0 x 31 x x x 28* x x xx x x x x x 4 x x x x x x 32 x x x  9* x x x x x x x x x 23  x x x x x x34 2 0 1 0 1 0 2  5* 0 2 0 0 0 0 1 3 8 5 0 2 39 3 0 3  2* 1 10  1 0 2 22 1 0 3 1 0 0 1 0 0

TABLE 18 Distribution of amino-acid types at varied residues in proteinsselected for plasmin binding from third library. Position A C D E F G HI K L M N P Q R S T V W Y 10 x x  7* 8 x x x x 0 x x 0 x x x x x x x x11 4 x  0* x x 0 x 0 x x x 0 x x x 2 7 2 x x 13 0 x x x x x x x x x x x15* x x 0 0 x x x 15 x x x x x x x x  2* x x x x x 13  x x x x x 16 10*x x x x 5 x x x x x x x x x x x x x x 17 x x x x x x x  0* 11  x 0 0 x x3 1 0 x x x 18 x x x x 14  x x 1 x x  x* x x x x x x x x x 19 0 0 8 5 01 0 0  0* 0 0 0 1 0 0 0 0 0 0 0 21 x 0 x x  5* x x x x 2 x x x x x x x x8 x 31 1 0 1  6* 0 0 1 0 0 0 0 2 0 1 0 0 3 0 0 0 32 4 0 0  1* 0 1 0 0 01 0 0 2 1 1 1 2 1 0 0 34 0 0 1 x 1 0 1  2* x 3 x 0 1 x 1 0 1 4 x 0 35 x0 x x x x x x x x x x x x x x x x 2 13* 39 x x x  5* x 8 x x 1 x x x x x1 x x x x x

TABLE 23 Specificity Results Obtained with KuDoms Displayed on gIIIp ofM13 Target KuDom Trypsin, 2 Displayed Plasmin Thrombin KallikreinTrypsin washes LACI-K1 1.0 1.0 1.0 1.0 1.0 QS4 52. 0.7 0.9 4.5 0.5 BPTI88. 1.1 1.7 0.3 0.8LACI-K1 phage for each Target was taken as unit binding and the otherdisplay phage are shown as relative binding. BPTI::III phage are noteasliy liberated from trypsin.

TABLE 24 Mat α S. cerevisiae expression vectors: Matα1 (Mfα8)

Matα2 (after introduction of a linker into StuI-cut DNA)

Matα-LACI-K1

We expect that Matα pre sequence is cleaved before GLU_(a) -ALA_(a)-

CITATIONS

-   ADEL86: Adelman et al., Blood (1986) 68(6)1280-1284.-   ANBA88: Anba et al., Biochimie (1988) 70(6)727-733.-   AUER88: Auerswald et al., Bio Chem Hoppe-Seyler (1988), 369    (Supplement):27-35.-   BANE90: Baneyx & Georgiou, J Bacteriol (1990) 172(1)491-494.-   BANE91: Baneyx & Georgiou, J Bacteriol (1991) 173(8)2696-2703.-   BROW91: Browne et al., GeneBank entry M74220.-   BROZ90: Broze et al., Biochemistry (1990) 29:7539-7546.-   COLM87: Colman et al., Editors, Hemostasis and Thrombosis, Second    Edition, 1987, J. B. Lippincott Company, Philadelphia, Pa.-   DENN94a: Dennis & Lazarus, J Biological Chem (1994) 269:22129-22136.-   DENN94b: Dennis & Lazarus, J Biological Chem (1994) 269:22137-22144.-   EIGE90: Eigenbrot et al., Protein Engineering (1990), 3(7)591-598.-   ELLI92: Ellis et al., Ann NY Acad Sci (1992) 667:13-31.-   FIDL94: Fidler & Ellis, Cell (1994) 79:185-188.-   FRAE89: Fraedrich et al., Thorac Cardiovasc Surg (1989) 37(2)89-91.-   GARD93: Gardell, Toxicol Pathol (1993) 21(2)190-8.-   GIRA89: Girard et al., Nature (1989), 338:518-20.-   GIRA91: Girard et al., J. BIOL. CHEM. (1991) 266:5036-5041.-   GREG93: Gregg et al., Bio/Technology (1993)11:905-910.-   HOOV93: Hoover et al., Biochemistry (1993) 32:10936-43.-   HYNE90: Hynes et al., Biochemistry (1990), 29:10018-10022.-   KIDO88: Kido et al., J Biol Chem (1988), 263:18104-7.-   KIDO90: Kido et al., Biochem & Biophys Res Comm (1990),    167(2)716-21.-   KURJ82: Kurjan and Herskowitz, Cell (1982) 30:933-943.-   LASK80: Laskowski & Kato, Ann Rev Biochem (1980), 49:593-626.-   LEAT91: Leatherbarrow & Salacinski, Biochemistry (1991)    30(44)10717-21.-   LOHM93: Lohmann & J Marshall, Refract Corneal Surg (1993) 9(4)300-2.-   LUCA83: Lucas et al., J Biological Chem (1983) 258(7)4249-56.-   MANN87: Mann & Foster, Chapter 10 of COLM87.-   MIYA85: Miyajima et al., Gene (1985) 37:155-161.-   NEUH89: Neuhaus et al., Lancet (1989) 2(8668)924-5.-   NOVO89: Novotny et al. J. BIOL. CHEM. (1989) 264:18832-18837.-   OREI94: O'Reilly et al., Cell (1994) 79:315-328.-   PARK86: Park & Tulinsky, Biochemistry (1986) 25(14)3977-3982.-   PUTT89: Putterman, Acta Chir Scand (1989) 155(6-7)367.-   ROBB87: Robbins, Chapter 21 of COLM87-   SCHE67: Schechter & Berger, Biochem Biophys Res Commun (1967)    27:157-162.-   SCHE68: Schechter & Berger, Biochem Biophys Res Commun (1969)    32:898-902.-   SCHN86: Schnabel et al., Biol Chem Hoppe-Seyler (1986), 367:1167-76.-   SHER89: Sheridan et al., Dis Colon Rectum (1989) 32(6)505-8.-   SPRE94: Sprecher et al., Proc Natl Acad Sci USA 91:3353-3357 (1994)-   VAND92: van Dijl et al., EMBO J (1992) 11(8)2819-2828.-   VARA83: Varadi & Patthy, Biochemistry (1983) 22:2440-2446.-   VARA84: Varadi & Patthy, Biochemistry (1984) 23:2108-2112.-   VEDV91: Vedvick et al., J Ind Microbiol (1991) 7:197-201.-   WAGN92: Wagner et al., Biochem Biophys Res Comm (1992)    186:1138-1145.-   WUNT88: Wun et al., J. BIOL. CHEM. (1988) 263:6001-6004.

1.-11. (canceled)
 12. A method of assaying a sample for plasmincomprising: contacting the sample with a polypeptide comprising anon-naturally occurring Kunitz domain that has the formula:Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Xaa10-Xaa11-Gly-Xaa13-Cys-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Arg-Trp-Xaa22-Xaa23-Asn-Ile-Phe-Thr-Arg-Gln-Cys-Xaa31-Xaa32-Phe-Xaa34-Xaa35-Gly-Gly-Cys-Xaa39-Xaa40-Asn-Gln-Xaa43-Arg-Xaa45-Glu-Ser-Leu-Glu-Glu-Cys-LysLysMet-Cys-Thr-Arg-Asp, wherein Xaa10 is selected from the group consistingof Asp, Glu, and Tyr; Xaa11 is selected from the group consisting ofThr, Ala, Ser, Val, and Asp; Xaa13 is selected from the group consistingof Pro, Leu, and Ala; Xaa15 is selected from the group consisting of Argand Lys; Xaa16 is selected from the group consisting of Ala and Gly;Xaa17 is selected from the group consisting of Arg, Lys, and Ser; Xaa18is selected from the group consisting of Phe and Ile; Xaa19 is selectedfrom the group consisting of Glu, Asp, Pro, Gly, Ser, and Ile; Xaa22 isselected from the group consisting of Tyr and Phe; Xaa23 is selectedfrom the group consisting of Tyr and Phe; Xaa31 is selected from thegroup consisting of Asp, Glu, Thr, Val, Gln, and Ala; Xaa32 is selectedfrom the group consisting of Thr, Ala, Glu, Pro, and Gln; Xaa34 isselected from the group consisting of Val, Ile, Thr, Leu, Phe, Tyr, His,Asp, Ala, and Ser; Xaa35 is selected from the group consisting of Tyrand Trp; Xaa39 is selected from the group consisting of Glu, Gly, Asp,Arg, Ala, Gln, Leu, Lys, and Met; Xaa40 is selected from the groupconsisting of Gly and Ala; Xaa43 is selected from the group consistingof Asn and Gly; and Xaa45 is selected from the group consisting of Pheand Tyr; and detecting the presence of a complex of said polypeptide andplasmin in the sample.
 13. The method according to claim 12, whereinsaid polypeptide comprises a Kunitz domain having the formula:Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Xaa10-Xaa11-Gly-Xaa13-Cys-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Arg-Trp-Xaa22-Xaa23-Asn-Ile-Phe-Thr-Arg-Gln-Cys-Xaa31-Xaa32-Phe-Xaa34-Xaa35-Gly-Gly-Cys-Xaa39-Xaa40-Asn-Gln-Xaa43-Arg-Xaa45-Glu-Ser-Leu-Glu-Glu-Cys-Lys-Lys-Met-Cys-Thr-Arg-Asp,wherein Xaa10 is selected from the group consisting of Asp and Glu;Xaa11 is selected from the group consisting of Thr, Ala, Ser, Val, andAsp; Xaa13 is selected from the group consisting of Pro, Leu, and Ala;Xaa15 is selected from the group consisting of Arg and Lys; Xaa16 isselected from the group consisting of Ala and Gly; Xaa17 is selectedfrom the group consisting of Arg, Lys, and Ser; Xaa18 is selected fromthe group consisting of Phe and Ile; Xaa19 is selected from the groupconsisting of Glu, Asp, Pro, Gly, Ser, and Ile; Xaa22 is Phe; Xaa23 isPhe; Xaa31 is selected from the group consisting of Asp, Glu, Thr, Val,Gln, and Ala; Xaa32 is selected from the group consisting of Thr, Ala,Glu, Pro, and Gln; Xaa34 is selected from the group consisting of Val,Ile, Thr, Leu, Phe, Tyr, His, Asp, Ala, and Ser; Xaa35 is selected fromthe group consisting of Tyr and Trp; Xaa39 is selected from the groupconsisting of Glu, Gly, Asp, Arg, Ala, Gln, Leu, Lys, and Met; Xaa40 isselected from the group consisting of Gly and Ala; Xaa43 is selectedfrom the group consisting of Asn and Gly; and Xaa45 is selected from thegroup consisting of Phe and Tyr.
 14. The method according to claim 12,wherein said polypeptide comprises a Kunitz domain having a sequenceselected from the group consisting of: (SEQ ID NO: 40)MHSFCAFKAETGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEE CKKMCTRD; (SEQ ID NO:41) MHSFCAFKAESGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRF ESLEECKKMCTRD; (SEQ IDNO: 43) MHSFCAFKAEGGPCRAKFQRWFFNIFTRQCEEFIYGGCEGNQNRFESLEE CKKMCTRD;(SEQ ID NO: 45) MHSFCAFKAETGPCRAKFDRWFFNIFTRQCEAEVYGGCGGNQNRFESLEECKKMCTRD; (SEQ ID NO: 46)MHSFCAFKAETGPCRAKFDRWFFNIFTRQCEPFVYGGCEGNQNRFESLEE CKKMCTRD; (SEQ ID NO:47) MHSFCAFKAETGPCRAKFDRWFFNIFTRQCNTFVYGGCGGNQNRFESLEE CKKMCTRD; (SEQ IDNO: 48) MHSFCAFKAETGPCRGKFDRWFFNIFTRQCQFVYGGCGGNQNRFESLEEC KKMCTRD; (SEQID NO: 49) MHSFCAFKAEVGPCRAKFDRWFFNIFTRQCHLFTYGGCGGNQNRFESLEE CKKMCTRD;(SEQ ID NO: 50) MHSFCAFKAETGPCRGKFDRWFFNIFTRQCAQFVYGGCEGNQNRFESLEECKKMCTRD; (SEQ ID NO: 51)MHSFCAFKAETGPCRGKFDRWFFNIFTRQCESFHYGGCKGNQNRFESLEE CKKMCTRD; (SEQ ID NO:55) MHSFCAFKADSGPCRARFDRWFFNIFTRQTRYPYGGCGGNQNRFESLEEC KKMCTRD; (SEQ IDNO: 58) MHSFCAFKAETGPCKGSFDRWFFNIFTRQCNVFRYGGCRGNQNRFESLEE CKKMCTRD;(SEQ ID NO: 117) MHSFCAFKAETGPCAARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEECKKMCTRD; (SEQ ID NO: 118)MHSFCAFKAETGPCGARFDRWFFNIFTRQCEEFIYGGCEGNQNPYESLEE CKKMCTRD; and (SEQ IDNO: 119) MHSFCAFKAETGPCNARFDRWFFNIFTRQCEAFIYGGCEGNQNRFESLEE CKKMCTRD.


15. The method according to claim 12, wherein said polypeptide comprisesa Kunitz domain having the sequence: (SEQ ID NO: 40)MHSFCAFKAETGPCRARFDRWFFNIFTRQCEEFIYGGCEGNQNRFESLEE CKKMCTRD.


16. A method of assaying a sample for plasmin comprising: contacting thesample with a polypeptide comprising a non-naturally occurring Kunitzdomain that has the formula:Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Xaa10-Xaa11-Gly-Xaa13-Cys-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Arg-Xaa21-Xaa22-Xaa23-Asn-Ile-Phe-Thr-Arg-Gln-Cys-Xaa31-Xaa32-Phe-Xaa34-Xaa35-Gly-Gly-Cys-Xaa39-Xaa40-Asn-Gln-Xaa43-Arg-Xaa45-Glu-Ser-Leu-Glu-Glu-Cys-Lys-Lys-Met-Cys-Thr-Arg-Asp,wherein Xaa10 is selected from the group consisting of Asp, Glu, andTyr; Xaa11 is selected from the group consisting of Thr, Ala, Ser, Val,and Asp; Xaa13 is selected from the group consisting of Pro, Leu, andAla; Xaa15 is selected from the group consisting of Arg and Lys; Xaa16is selected from the group consisting of Ala and Gly; Xaa17 is selectedfrom the group consisting of Arg, Lys, and Ser; Xaa18 is selected fromthe group consisting of Phe and Ile; Xaa19 is selected from the groupconsisting of Glu, Asp, Pro, Gly, Ser, and Ile; Xaa21 is selected fromthe group consisting of Phe, Trp, and Tyr; Xaa22 is selected from thegroup consisting of Tyr and Phe; Xaa23 is selected from the groupconsisting of Tyr and Phe; Xaa31-Xaa32 is selected from the groupconsisting of Glu-Gln, Gln-Gln, and Gln-Glu; Xaa34 is selected from thegroup consisting of Val, Ile, Thr, Leu, Phe, Tyr, His, Asp, Ala, andSer; Xaa35 is selected from the group consisting of Tyr and Trp; Xaa39is selected from the group consisting of Glu, Gly, Asp, Arg, Ala, Gln,Leu, Lys, Phe, Asn, His and Met; Xaa40 is selected from the groupconsisting of Gly and Ala; Xaa43 is selected from the group consistingof Asn and Gly; and Xaa45 is selected from the group consisting of Pheand Tyr.
 17. The method according to claim 16, wherein said polypeptidecomprises a Kunitz domain having the formula:Met-His-Ser-Phe-Cys-Ala-Phe-Lys-Ala-Asp-Asp-Gly-Pro-Cys-Lys-Ala-Arg-Phe-Glu-Arg-Phe-Phe-Phe-Asn-Ile-Phe-Thr-Arg-Gln-Cys-Xaa31-Xaa32-Phe-Xaa34-Tyr-Gly-Gly-Cys-Xaa39-Gly-Asn-Gln-Asn-Arg-Phe-Glu-Ser-Leu-Glu-Glu-Lys-Met-Cys-Thr-Arg-Asp,wherein Xaa31-Xaa32 is selected from the group consisting of Glu-Gln,Gln-Gln, and Gln-Glu; Xaa34 is selected from the group consisting ofVal, Ile, Thr, Leu, Phe, Tyr, His, Asp, Ala, and Ser; and Xaa39 isselected from the group consisting of Glu, Gly, Asp, Arg, Ala, Gln, Leu,Lys, Phe, Asn, His and Met.
 18. The method according to claim 16,wherein said polypeptide comprises a Kunitz domain having a sequenceselected from the group consisting of: (SEQ ID NO: 3)MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFTYGGCRGNQNRFESLEE CKKMCTRD; (SEQ ID NO:4) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFYYGGCDGNQNRFESLEE CKKMCTRD; (SEQ IDNO: 5) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFHYGGCDGNQNRFESLEE CKKMCTRD; (SEQID NO: 6) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFDYGGCAGNQNRFESLEE CKKMCTRD;(SEQ ID NO: 7) MHSFCAFKADDGPCKARFERFFFNIFTRQCQEFRYGGCDGNQNRFESLEECKKMCTRD; (SEQ ID NO: 8)MHSFCAFKADDGPCKARFERFFFNIFTRQCQQFYYGGCQGNQNRFESLEE CKKMCTRD; (SEQ ID NO:10) MHSFCAFKADDGPCKARFERFFFNIFTRQCQQFVYGGCGGNQNRFESLEE CKKMCTRD; (SEQ IDNO: 11) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFTYGGCGGNQNRFESLEE CKKMCTRD;(SEQ ID NO: 13) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFIYGGCQGNQNRFESLEECKKMCTRD; (SEQ ID NO: 14)MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFIYGGCGGNQNRFESLEE CKKMCTRD; (SEQ ID NO:15) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFIYGGCFGNQNRFESLEE CKKMCTRD; (SEQ IDNO: 16) MHSFCAFKADDGPCKARFERFFFNIFTRQCQQFHYGGCEGNQNRFESLEE CKKMCTRD;(SEQ ID NO: 17) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFVYGGCAGNQNRFESLEECKKMCTRD; (SEQ ID NO: 18)MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFLYGGCGGNQNRFESLEE CKKMCTRD; (SEQ ID NO:19) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFIYGGCGGNQNRFESLEE CKKMCTRD; (SEQ IDNO: 20) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFVYGGCGGNQNRFESLEE CKKMCTRD;(SEQ ID NO: 23) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFTYGGCMGNQNRFESLEECKKMCTRD; (SEQ ID NO: 24)MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFSYGGCGGNQNRFESLEE CKKMCTRD; (SEQ ID NO:26) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFSYGGCQGNQNRFESLEE CKKMCTRD; (SEQ IDNO: 27) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFAYGGCAGNQNRFESLEE CKKMCTRD;(SEQ ID NO: 28) MHSFCAFKADDGPCKAFERFFFNIFTRQCEQFIYGGCVGNQNRFESLEECKLKMCTRD; (SEQ ID NO: 31)MHSFCAFKADDGPCKASFERFFFNIFTRQCEQFTYGGCNGNQNRFESLEE CKKMCTRD; (SEQ ID NO:33) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFFYGGCHGNQNRFESLEE CKKMCTRD; (SEQ IDNO: 34) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFTYGGCGGNQNRFESLEE CKKMCTRD;(SEQ ID NO: 35) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFTYGGCMGNQNRFESLEECKKMCTRD; (SEQ ID NO: 36)MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFTYGGCGGNQNRFESLEE CKKMCTRD; and (SEQ IDNO: 37) MHSFCAFKADDGPCKARFERFFFNIFTRQCEQFVYGGCGGNQNRFESLEE CKKMCTRD.


19. The method according to claim 18, wherein said polypeptide comprisesa Kunitz domain having the sequence: (SEQ ID NO: 4)MHSFCAFKADDGPCKARFEREFFNIFTRQCEQFYYGGCDGNQNRFESLEE CKKMCTRD.


20. The method according to claim 12, wherein said polypeptide has aK_(i) for human plasmin of 100 pM or less.
 21. The method according toclaim 16, wherein said polypeptide has a K_(i) for human plasmin of 100pM or less.
 22. The method according to claim 12, wherein saidpolypeptide further comprises one or more amino acids upstream of theKunitz domain.
 23. The method according to claim 22, wherein the one ormore amino acids are involved with processing by a recombinant hostcell.
 24. The method according to claim 15, wherein said polypeptidefurther comprises one or more amino acids upstream of the Kunitz domain.25. The method according to claim 24, wherein the one or more aminoacids are involved with processing by a recombinant host cell.
 26. Themethod according to claim 16, wherein said polypeptide further comprisesone or more amino acids upstream of the Kunitz domain.
 27. The methodaccording to claim 26, wherein the one or more amino acids are involvedwith processing by a recombinant host cell.
 28. The method according toclaim 19, wherein said polypeptide further comprises one or more aminoacids upstream of the Kunitz domain.
 29. The method according to claim12, wherein the polypeptide comprises a label
 30. The method accordingto claim 29, wherein the label is selected from the group consisting ofa radioisotope, a fluorophore, an enzyme, a co-enzyme, an enzymesubstrate, an electron-dense compound and an agglutinable particle. 31.The method according to claim 16, wherein the polypeptide comprises alabel
 32. The method according to claim 31, wherein the label isselected from the group consisting of a radioisotope, a fluorophore, anenzyme, a co-enzyme, an enzyme substrate, an electron-dense compound andan agglutinable particle.